Device and method for separating plasma from a blood sample

Abstract

The present invention relates to a microfluidic device and a method for separating plasma from a blood sample, and to the use of same. The device allows the processing of millimetric blood samples, improved plasma extraction, and the extraction of platelet-enriched high-purity plasma with a low content of activated platelets.

Description

[0001] DESCRIPTION

[0002] DEVICE FOR SEPARATING PLASMA FROM A BLOOD SAMPLE

[0003] OBJECT OF THE INVENTION

[0004] The present invention relates to a microfluidic device and a method for separating plasma from a blood sample, as well as to the use thereof. The device allows the processing of blood samples on the order of milliliters, an improvement in plasma extraction, and the extraction of high-purity plasma enriched with platelets and with a low content of activated platelets.

[0005] BACKGROUND OF THE INVENTION

[0006] Microfluidic devices have revolutionized various scientific and technological fields by enabling the precise manipulation of small volumes of fluids, typically in the microliter to nanoliter range. These systems are based on the microfabrication of channels and chambers within materials such as silicon, glass, or polymers, using advanced techniques like photolithography, soft molding, and 3D printing.

[0007] The miniaturization offered by microfluidic devices has enabled their integration into laboratory platforms in chip form, which has led to the automation of analytical processes that previously required bulky equipment and manual steps.

[0008] One of the most significant applications of microfluidics is in the biomedical field, where it is used for the analysis and processing of biological fluids such as blood, urine, and semen. Among these applications, the separation of plasma from blood is particularly crucial, since plasma is the liquid fraction of blood that contains vital components such as proteins, hormones, electrolytes, and metabolites, which are essential for disease diagnosis, treatment monitoring, and clinical research studies.

[0009] In whole blood, plasma makes up the majority of the sample volume, while the remainder consists of cells, primarily erythrocytes (red blood cells), leukocytes (white blood cells), and platelets. Efficient separation of these cells from the plasma is essential for obtaining samples that can be accurately analyzed later. Conventional plasma separation methods, such as centrifugation, while effective, require specialized equipment, considerable processing time, and may be impractical or have reduced efficiency in some applications.

[0010] Microfluidic devices for plasma separation utilize a variety of physical and mechanical principles to perform this task. Among the most common techniques are: filtration separation, capillary force separation, velocity gradient separation, centrifugation separation, electric field separation, etc.

[0011] Despite advances in the design and manufacture of these devices, separation efficiency, the purity of the plasma obtained, and the concentration of platelets in the plasma are areas where significant challenges remain.

[0012] In particular, centrifugation-based devices for isolating platelet-rich plasma can lead to blood contamination, low reproducibility of the separation, and unwanted platelet activation of up to 50%. In this respect, microfluidic devices offer less platelet activation, better portability, and greater automation of platelet recovery compared to traditional methods. Simple microfluidic designs that utilize gravity can achieve very pure separation of plasma from whole blood. However, these devices separate limited plasma volumes due to their single-trench designs.

[0013] Therefore, there is a need in this field of technology for a microfluidic device with multiple trenches that positively impacts the quantity and purity of plasma collected and the platelet count obtained, with less platelet activation. A microfluidic device is also needed that offers these advantages and is capable of processing whole blood volumes on the order of milliliters.

[0014] DESCRIPTION OF THE INVENTION The present invention proposes a solution to the aforementioned problems by means of a device for obtaining platelet-enriched plasma from a blood sample according to claims 1, 3 and 5, a method for obtaining platelet-enriched plasma from a blood sample according to claim 12 and the use of the device for obtaining platelet-enriched plasma from a blood sample according to claim 16. Preferred embodiments of the invention are defined in the dependent claims.

[0015] In a first inventive aspect, the present invention provides a microfluidic device for obtaining platelet-enriched plasma from a blood sample, wherein the microfluidic device comprises:

[0016] - a channel comprising an inlet and an outlet, an upper and a lower part, and a defined height H between the upper and lower parts of the channel, the inlet being configured for the introduction of a blood sample, the outlet being configured to fluidly couple a pump configured to extract plasma from the channel, and comprising a flow direction from its inlet to its outlet;

[0017] - a plurality of cavities distributed along at least a portion of the channel, each cavity comprising a lower surface and an upper surface, and where a microchannel is formed between the upper part of the channel and the upper surface of the plurality of cavities; where each cavity of the plurality of cavities is elongated in a direction perpendicular to the flow direction, and where each cavity comprises a height X; a width A, said width A being between 0.5 and 2.5 mm; a length L, said length L being between 1.5 and 4.5 mm; between any two adjacent cavities there is a separation distance S between them, said distance S being between 0.5 and 1.5 mm; the channel comprises a height H being between 1 and 3 mm; and a length T, said length T being between 30 and 90 mm; and the microchannel comprises a depth C, said depth C being between 100 and 400 pm;and wherein each of the cavities comprises a hydrophilic coating on its lower surface and the height X of each of the cavities is defined by the relation X = H - C.;

[0018] The term “microfluidics” refers to the design, fabrication, and formulation of designs and processes that deal with fluid volumes, typically on the order of nanoliters, microliters, or milliliters. A “microfluidic device,” as used herein, refers to any device or system that allows the precise control and manipulation of fluids that are geometrically confined to structures in which at least one dimension (width, length, height) is less than 1 millimeter.

[0019] The term “plasma” or the expression “blood plasma,” in the present invention, refers to the non-cellular fluid portion of human or animal blood that exists before coagulation. It is composed of 90% water, 7% protein, and the remaining 3% consists of fat, glucose, vitamins, hormones, oxygen, carbon dioxide, and nitrogen, as well as metabolic waste products such as uric acid. It is the main component of blood, representing approximately 55% of the total blood volume, while the remaining 45% corresponds to the formed elements. The viscosity of blood plasma is 1.5 times that of water.

[0020] The main plasma proteins are the following:

[0021] - albumin, which also participates in lipid transport;

[0022] - globulins, which are mainly related to the body's defense mechanisms (for example, immunoglobulins) or to the transport of iron (transferrin);

[0023] - fibrinogen, a protein that is essential for blood clotting;

[0024] - prothrombin, a protein that participates in the coagulation process; and

[0025] - low and high density lipoproteins (LDL and HDL, respectively) that participate in the transport of cholesterol.

[0026] The set of plasma proteins, also called "plasma factors", performs the functions, among others, of:

[0027] - to maintain plasma volume and blood volume, - to protect and maintain blood pH stability,

[0028] - participate in blood viscosity, and therefore contribute minimally to peripheral vascular resistance and vascular pressure (blood pressure), and

[0029] - intervene in the concentration of electrochemical equilibrium ions (called the Donnan effect).

[0030] The term “plasma collection” or “plasma separation,” as used herein, refers to separating or extracting plasma from a whole blood sample. The terms “blood sample” and “whole blood sample” are defined below.

[0031] The term “platelets” or “thrombocytes,” as used herein, refers to small (2–3 micrometers in diameter), oval-shaped, anucleate cells generated in the bone marrow from the cytoplasmic fragmentation of megakaryocytes. Platelets circulate in the blood of all mammals and are involved in hemostasis, participating in the formation of blood clots or thrombi. Platelets release a large number of growth factors and / or cytokines, including platelet-derived growth factor (PDGF), a potent chemotactic agent, and transforming growth factor-beta (TGF-β), which stimulates the formation of the extracellular matrix.Depending on certain conditions, platelets also synthesize many other molecules, as previously mentioned, that are essential to the inflammatory process and the tissue repair and regeneration process. These growth factors or molecules have been shown to play an important role in the regeneration and repair of connective tissue.Other proteins and growth factors produced by platelets and associated inflammatory, reparative, and regenerative processes include basic fibroblast growth factor (BFGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), epithelial growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), interleukin-8, keratinocyte growth factor, and connective tissue growth factor. The local application of all these growth factors, molecules, and proteins produced by platelets participates in and accelerates the healing process of various injuries.Platelets also release large amounts of thrombin and adenosine diphosphate ADP, calcium, thromboxane A2, PF4, PAI-1, fibrinogen, fibronectin, thrombomodulin, FV, FXIII, RANTES, thrombospondin, WWF PF-3, serotonin, hydrolytic enzymes, etc.

[0032] The term “platelet-rich plasma”, as used herein, refers to plasma with a high platelet content.

[0033] The term “high-purity plasma,” as used herein, refers to plasma that is free of red blood cells and free of white blood cells, or, in other words, plasma that contains no red blood cells or white blood cells, or contains very low amounts of both. In a particular realization, the plasma obtained contains no red blood cells and no white blood cells, or contains less than 20%, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less of each of the red and white blood cells relative to the baseline concentration of red and white blood cells in a whole blood sample. The terms “red blood cells”, “white blood cells” and “whole blood sample” are defined below.

[0034] The term “platelet activation” refers hereto to the process by which platelets present in plasma aggregate to form a clot and promote hemostasis and healing through, among other processes, the secretion of growth factors or intercellular mediators from cytoplasmic granules. In a particular embodiment, the plasma obtained contains no activated platelets, or contains less than 20%, less than 18%, less than 16%, less than 14%, less than 12%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less, of activated platelets relative to the total platelet concentration in the plasma obtained.

[0035] The term “blood,” as used in this description, refers to a tissue fluid that circulates through the capillaries, veins, and arteries of all vertebrates and invertebrates. Its characteristic red color is due to the presence of the hemoglobin pigment contained in erythrocytes (red blood cells). It is a type of specialized connective tissue with a liquid colloidal matrix and a complex composition. It comprises a solid phase, formed by formed elements (including leukocytes, erythrocytes, and platelets), and a liquid phase, represented by blood plasma. Its main function is the logistics of distribution and systemic integration, and its containment within the blood vessels (vascular space) allows for its distribution (blood circulation) throughout most of the body. The term “peripheral blood” refers to the volume of blood circulating away from the heart, that is, the blood that circulates throughout the body.

[0036] The term “erythrocytes” or “red blood cells” or “RBCs,” as used in this description, refers to the blood corpuscles responsible for transporting oxygen and carbon dioxide in the blood. They are called corpuscles because they lack a nucleus and organelles (only in mammals), and therefore cannot be strictly considered cells. They contain some enzymatic pathways, and their cytoplasm is almost entirely occupied by hemoglobin, a protein responsible for oxygen transport. The plasma membrane of erythrocytes contains glycoproteins (CD) that define the different blood groups and other cell identifiers. Erythrocytes are disc-shaped, biconcave, and depressed in the center, which increases the effective surface area of ​​the membrane. Mature erythrocytes lack a nucleus, because it is expelled in the last stage of maturation in the bone marrow before entering the bloodstream.

[0037] The term “leukocytes” or “white blood cells” or “WBCs,” as used in this description, refers to blood cells that are part of the cellular effectors of the immune system. They are migratory cells that use the blood as a vehicle to access different parts of the body. Leukocytes are responsible for destroying infectious agents and infected cells, and they also secrete protective substances such as antibodies, which fight infections. Based on the microscopic characteristics of their cytoplasm (staining) and nucleus (morphology), they are divided into:

[0038] - granulocytes and polymorphonuclear cells (neutrophils, basophils, and eosinophils) and have a polymorphic nucleus and numerous granules in the cytoplasm, with differential staining according to cell types; and

[0039] - agranulocytes or mononuclear cells (lymphocytes and monocytes): lack cytoplasmic granules and have a round nucleus.

[0040] The term “cells,” as used herein, refers to both red and white blood cells, as described above. The term “sample” or “blood sample,” as used herein, refers to blood from a subject, obtained by any method known to a person skilled in the art that is suitable for use in the microfluidic device of the present invention or for carrying out any of the methods provided by the present invention. In a particular embodiment, such a sample is a peripheral blood sample. In a particular embodiment, the blood sample is a “whole blood sample” or a “complete blood sample.”The term “whole blood sample” or “complete blood sample,” as understood in the invention, is a blood sample already obtained (i.e., previously drawn from a subject, preferably a human subject) that has not been treated to remove insoluble blood components. Therefore, a whole blood sample contains insoluble components. Preferably, the whole blood sample is a peripheral blood sample. Preferably, the whole blood sample is collected in extraction tubes containing an anticoagulant, such as sodium citrate.

[0041] The blood sample is usually drawn by puncture of an artery or vein, typically a vein on the inside of the elbow or the back of the hand, and the blood sample is collected in a sealed vial or syringe. A capillary puncture, usually from the heel or the distal phalanges of the fingers, can be performed for analysis using a micromethod.

[0042] The term “subject,” as used herein, refers to any animal with circulatory blood, preferably a mammal, more preferably a primate, and still more preferably a human being, male or female, of any age or race. Furthermore, the subject may be a healthy individual or a subject diagnosed with any pathology or disease.

[0043] The microfluidic device of the first inventive aspect of the invention allows for obtaining platelet-rich plasma from a blood sample thanks to a plurality of cavities with specific dimensions that give the device the capacity to process blood samples on the order of milliliters. The microfluidic device is based on a sedimentation process; that is, gravity acts on the cells present in a blood sample introduced into the device and, in combination with the plurality of cavities and the difference in densities between each component of the blood sample, allows for the extraction of plasma from the blood sample.

[0044] Furthermore, under operating conditions, plasma is extracted from the channel by means of a pump coupled to the device's outlet. The resulting plasma is highly pure and enriched with platelets. Additionally, thanks to the passive sedimentation effect, platelets experience less stress, resulting in improved platelet integrity in the extracted plasma and reduced platelet activation compared to conventional methods such as centrifugation. The advantage of extracting high-quality, platelet-enriched plasma with reduced activation using the invention's device is that it preserves the platelet-related effects, as platelets are present at a high concentration and exhibit reduced activation in the plasma extracted from the blood sample.Specifically, once the extracted plasma is injected into a subject, these unactivated platelets will be activated and aggregate to give rise to the formation of the blood clot and promote hemostasis and healing through, among other processes, the secretion of growth factors or intercellular mediators from the cytoplasmic granules.

[0045] Advantageously, the platelet-rich plasma extracted using the microfluidic device of the invention can be used for numerous clinical and research applications. For example, in research, it can be used to study platelet-related cardiovascular diseases, diabetes, cancer, immunity, inflammation, sepsis, etc., which often require platelet isolation for analysis. Furthermore, hospitals and clinics need the separation and preparation of platelet-rich plasma for subsequent transfusions or other applications, such as wound healing, bone and tissue regeneration, dental implants, skin rejuvenation, hair restoration, fertility treatments, etc.

[0046] The device of the first inventive aspect comprises a plurality of elongated cavities oriented perpendicular to the flow direction. The combination of the number of cavities, their shape through their specific dimensions, and the sedimentation phenomenon allows for the extraction of platelet-enriched plasma and ensures that said plasma is free of globules, as these globules are trapped within the plurality of cavities of the device. The microfluidic device also comprises a channel with an upper and a lower portion. A height H is defined between the upper and lower portions of the channel. Furthermore, the plurality of cavities comprises a lower surface and an upper surface. The lower surface is delimited by the lower portion of the channel and coincides with the bottom of each cavity.The upper surface is delimited by the portion defined by the separation distance S between two adjacent cavities; that is, the upper part of the plurality of cavities is a discontinuous zone that can be represented by the union of all the separation zones between two adjacent cavities. The discontinuous zone is substantially parallel to the lower surface.

[0047] In the device of the invention, a microchannel of depth C is formed between the top of the channel and the upper surface of the plurality of cavities. Thanks to the dimensions of the channel height H and the microchannel depth C, the height X of each cavity can be calculated using the relation X = H - C.

[0048] Along the channel of the device of the invention, and in operating mode, the blood sample contains progressively fewer blood cells as it approaches the channel outlet. That is, the cavities closer to the channel inlet trap more blood cells than the cavities closer to the outlet. Put another way, the multiple cavities act as a passive and progressive filter that, in combination with sedimentation, retains the blood cells and allows for the extraction of high-purity plasma—that is, plasma free of blood cells and enriched with platelets—through the channel outlet.

[0049] Furthermore, the device of the first inventive aspect of the invention comprises a hydrophilic coating on the lower surface of each of its cavities. The hydrophilic coating prevents the formation of bubbles within the device channel, particularly within the cavities, and facilitates the movement of the blood sample within the channel in the direction of flow. In one particular embodiment, the hydrophilic coating comprises a material selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polydopamine, and polyacrylic acid. In another particular embodiment, the hydrophilic coating is adhesive.

[0050] The device of the invention therefore allows the processing of blood samples of the order of milliliters, thanks to its specific dimensions and the shape of its cavities, passively based on the sedimentation of the globules within the same cavities so that platelet-enriched plasma can be extracted through the outlet of the device.

[0051] In one embodiment, the microfluidic device comprises a plurality of layers, preferably at least one layer comprising acrylic, i.e., polymethyl methacrylate (PMMA). In a particular embodiment, the device further comprises a transparent, pressure-sensitive coating.

[0052] In an embodiment of the first inventive aspect, the height H of the channel is 2 mm; the width A of each of the cavities is 1.5 mm; and the length L of each of the cavities is 3 mm.

[0053] In an embodiment of the first inventive aspect, the plurality of cavities comprises between 10 and 80 cavities, preferably between 25 and 65 trenches, more preferably 45 trenches.

[0054] Advantageously, in a particular embodiment of the first inventive aspect, the optimal number of trenches is 45. This allows for optimal device sizing while also ensuring the correct extraction of platelet-rich plasma from the blood sample. In other words, the device is designed to be compact but with enough trenches to trap all the blood cells in the sample, allowing only plasma to be extracted through the device's outlet. On the one hand, an expert in the field would understand that including more trenches would not improve plasma extraction but would only add dead space within the microfluidic device. On the other hand, fewer than 45 trenches could compromise the purity of the extracted plasma, meaning it might contain a certain number of blood cells.

[0055] In a second inventive aspect, the invention provides a microfluidic device for obtaining platelet-enriched plasma from a blood sample, wherein the microfluidic device comprises:

[0056] - a channel comprising an inlet and an outlet, an upper and a lower part, and a defined height H between the upper and lower parts of the channel, the inlet being configured for the introduction of a blood sample, the outlet being configured to fluidly couple a pump configured to extract plasma from the channel, and comprising a flow direction from its inlet to its outlet;

[0057] - a plurality of cavities distributed along at least a portion of the channel, each cavity comprising a lower surface and an upper surface, and where a microchannel is formed between the upper part of the channel and the upper surface of the plurality of cavities; where each cavity of the plurality of cavities is circular in shape, and where each cavity comprises a height X; a diameter D, said diameter D being between 1.5 and 4.5 mm; a separation distance S between any two adjacent cavities, said distance S being between 0.5 and 1.5 mm; the channel comprising a height H being between 1 and 3 mm; and a length T, said length T being between 30 and 90 mm; and the microchannel comprising a depth C, said depth C being between 100 and 400 mm;and wherein each of the cavities comprises a hydrophilic coating on its lower surface and the height X of each of the cavities is defined by the relation X = H - C.;

[0058] All the definitions described above in relation to the first inventive aspect are equally applicable to the second inventive aspect and its particular embodiments.

[0059] The microfluidic device of the second inventive aspect of the invention allows for obtaining platelet-rich plasma from a blood sample thanks to a plurality of cavities with specific dimensions that give the device the capacity to process blood samples on the order of milliliters. The microfluidic device is based on a sedimentation process; that is, gravity acts on the cells present in a blood sample introduced into the device and, in combination with the plurality of cavities and the difference in densities between each component of the blood sample, allows for the extraction of plasma from the blood sample.

[0060] Furthermore, under operating conditions, plasma is extracted from the channel by means of a pump coupled to the device's outlet. The resulting plasma is highly pure and enriched with platelets. Additionally, thanks to the passive sedimentation effect, the extracted plasma exhibits better platelet integrity and less platelet activation compared to conventional methods such as centrifugation. The advantage of extracting high-quality, platelet-enriched plasma with reduced activation using the invention's device is that it preserves the platelet-related benefits, as the platelets are present at a high concentration and exhibit less activation in the plasma extracted from the blood sample.Specifically, once the extracted plasma is injected into a subject, these unactivated platelets will be activated and aggregate to give rise to the formation of the blood clot and promote hemostasis and healing through, among other processes, the secretion of growth factors or intercellular mediators from the cytoplasmic granules.

[0061] Advantageously, platelet-rich plasma extracted using the microfluidic device of the invention can be used for numerous clinical and research applications. For example, in research, it can be used to study platelet-related cardiovascular diseases, diabetes, cancer, immunity, inflammation, sepsis, etc., which often require platelet isolation for analysis. Furthermore, hospitals and clinics need the separation and preparation of platelet-rich plasma for subsequent transfusions or other applications, such as wound healing, bone and tissue regeneration, dental implants, skin rejuvenation, hair restoration, fertility treatments, etc.

[0062] The device of the second inventive aspect comprises a plurality of circular cavities. The combination of the number of cavities, their shape through their specific dimensions, and the sedimentation phenomenon allows for the extraction of platelet-enriched plasma and ensures that said plasma does not contain globules, as these globules are trapped within the plurality of circular cavities of the device. The microfluidic device also comprises a channel with an upper and a lower portion. A height H is defined between the upper and lower portions of the channel. Furthermore, the plurality of cavities comprises a lower surface and an upper surface. The lower surface is delimited by the lower portion of the channel and coincides with the bottom of each cavity.The upper surface is delimited by the portion defined by the separation distance S between two adjacent cavities; that is, the upper part of the plurality of cavities is a discontinuous zone that can be represented by the union of all the separation zones between two adjacent cavities. The discontinuous zone is substantially parallel to the lower surface.

[0063] In the device of the invention, a microchannel of depth C is formed between the top of the channel and the upper surface of the plurality of cavities. Thanks to the dimensions of the channel height H and the microchannel depth C, the height X of each cavity can be calculated using the relation X = H - C.

[0064] Along the channel of the device of the invention, in operating mode, the blood sample contains progressively fewer blood cells as it approaches the channel outlet. That is, the cavities closer to the channel inlet trap more blood cells than the cavities closer to the outlet. Put another way, the multiple cavities act as a passive and progressive filter that retains blood cells in combination with sedimentation, allowing for the extraction of high-purity plasma—that is, plasma free of blood cells and enriched with platelets—through the channel outlet.

[0065] Furthermore, the device of the second inventive aspect of the invention comprises a hydrophilic coating on the lower surface of each of its cavities. The hydrophilic coating prevents the formation of bubbles within the device channel, particularly within the cavities, and facilitates the movement of the blood sample within the channel in the direction of flow.

[0066] The device of the invention therefore allows the processing of blood samples on the order of milliliters, thanks to its specific dimensions and the shape of its cavities, passively based on the sedimentation of the blood cells within these cavities, so that platelet-rich plasma can be extracted through the device's outlet. In one embodiment, the microfluidic device comprises a plurality of layers, preferably at least one layer comprising acrylic, i.e., polymethyl methacrylate (PMMA). In a particular embodiment, the device further comprises a transparent, pressure-sensitive coating.

[0067] In an embodiment of the second inventive aspect, the height H of the channel is 2 mm; and the diameter D of each of the cavities is 3 mm.

[0068] In an embodiment of the second inventive aspect, the plurality of cavities comprises between 10 and 60 cavities, preferably between 20 and 45 trenches, more preferably 30 trenches.

[0069] Advantageously, in a particular embodiment of the second inventive aspect, the optimal number of trenches is 30. This allows for optimal device sizing and ensures the correct extraction of platelet-rich plasma from the blood sample. In other words, the device is designed with a compact size but with enough trenches to trap all the blood cells in the sample, allowing only plasma to be extracted through the device's outlet. On the one hand, an expert in the field would understand that including more trenches would not improve plasma extraction but would only add dead space within the microfluidic device. On the other hand, fewer than 30 trenches could compromise the purity of the extracted plasma, meaning it might contain a certain number of blood cells.

[0070] In a third inventive aspect, the invention provides a microfluidic device for obtaining platelet-enriched plasma from a blood sample, wherein the microfluidic device comprises:

[0071] - a channel comprising an inlet and an outlet, an upper and a lower part, and a defined height H between the upper and lower parts of the channel, the inlet being configured for the introduction of a blood sample, the outlet being configured to fluidically couple a pump configured to extract plasma from the channel, and comprising a flow direction from its inlet to its outlet;

[0072] - a plurality of cavities distributed along at least a portion of the channel, each cavity comprising a lower surface and an upper surface, and where a microchannel is formed between the upper part of the channel and the upper surface of the plurality of cavities; where each cavity of the plurality of cavities is elongated in the flow direction, where each cavity comprises a height X; a width A, said width A being between 0.5 and 4.5 mm; a length L, said length L being between 20 and 60 mm; the channel comprises a height H being between 1 and 3 mm; and a length T, said length T being between 30 and 90 mm; the microchannel comprises a depth C, said depth C being between 100 and 400 mm; and where each cavity comprises a hydrophilic coating on its lower surface and the height X of each cavity is defined by the relation X = H - C.

[0073] All the definitions described above in relation to the first inventive aspect are equally applicable to the third inventive aspect and its particular embodiments.

[0074] The microfluidic device of the third inventive aspect of the invention allows for obtaining platelet-enriched plasma from a blood sample thanks to a plurality of cavities with specific dimensions, giving the device the capacity to process blood samples on the order of milliliters. The microfluidic device is based on a sedimentation process; that is, gravity acts on the cells present in a blood sample introduced into the device, and, in combination with the plurality of cavities and the difference in densities between each component of the blood sample, allows for the extraction of plasma from the blood sample. Furthermore, under operating conditions, the plasma is extracted from the channel by means of a pump coupled to the device's outlet, where the obtained plasma has high purity and is enriched with platelets.Furthermore, thanks to the passive effect of sedimentation, there is better platelet integrity in the extracted plasma and less platelet activation compared to conventional methods such as centrifugation. The advantage of extracting high-quality platelet-enriched plasma with reduced activation using the device of the invention is that it preserves the platelet-related effects, as platelets are present in a high concentration and with less activation in the plasma extracted from the blood sample. Specifically, once the extracted plasma is injected into a subject, these inactive platelets will be activated and aggregate, leading to blood clot formation and promoting hemostasis and wound healing through, among other processes, the secretion of growth factors or intercellular mediators from the cytoplasmic granules.

[0075] Advantageously, the platelet-rich plasma extracted using the microfluidic device of the invention can be used for numerous clinical and research applications. For example, in research, it can be used to study platelet-related cardiovascular diseases, diabetes, cancer, immunity, inflammation, sepsis, etc., which often require platelet isolation for analysis. Furthermore, hospitals and clinics need the separation and preparation of platelet-rich plasma for subsequent transfusions or other applications, such as wound healing, bone and tissue regeneration, dental implants, skin rejuvenation, hair restoration, fertility treatments, etc.

[0076] The device of the third inventive aspect comprises a plurality of cavities with an elongated shape in the flow direction. The combination of the number of cavities, their shape through their specific dimensions, and the sedimentation phenomenon allows for the extraction of platelet-enriched plasma and ensures that said plasma does not contain globules, since these globules are trapped within the plurality of cavities of the device.

[0077] The microfluidic device also comprises a channel with an upper and a lower portion. A height H is defined between the upper and lower portions of the channel. Furthermore, the plurality of cavities comprises a lower surface and an upper surface. The lower surface is bounded by the bottom of the channel and coincides with the bottom of each cavity. The upper surface is bounded by the portion defined by the separation distance S between two adjacent cavities; that is, the upper portion of the plurality of cavities is a discontinuous zone that can be represented by the union of all the separation zones between two adjacent cavities. This discontinuous zone is substantially parallel to the lower surface.

[0078] In the device of the invention, a microchannel of depth C is formed between the upper part of the channel and the upper surface of the plurality of cavities. Thanks to the dimensions of the channel height H and the microchannel depth C, the height X of each cavity can be calculated using the relation X = H - C.

[0079] Along the channel of the device of the invention, and in operating mode, the blood sample contains progressively fewer blood cells as it approaches the channel outlet. That is, the cavities closer to the channel inlet trap more blood cells than the cavities closer to the outlet. Put another way, the multiple cavities act as a passive and progressive filter that, in combination with sedimentation, retains the blood cells and allows for the extraction of high-purity plasma—that is, plasma free of blood cells and enriched with platelets—through the channel outlet.

[0080] Furthermore, the device of the third inventive aspect of the invention comprises a hydrophilic coating on the lower surface of each of its cavities. The hydrophilic coating prevents the formation of bubbles within the device channel, particularly within the cavities, and facilitates the movement of the blood sample within the channel in the direction of flow.

[0081] The device of the invention therefore allows the processing of blood samples of the order of milliliters, thanks to its specific dimensions and the shape of its cavities, passively based on the sedimentation of the globules within the same cavities so that platelet-enriched plasma can be extracted through the outlet of the device.

[0082] In one embodiment, the microfluidic device comprises a plurality of layers, preferably at least one layer comprising acrylic, i.e., polymethyl methacrylate (PMMA). In a particular embodiment, the device further comprises a transparent, pressure-sensitive coating.

[0083] In an embodiment of the third inventive aspect, the height H of the channel is 2 mm; the width A of each of the cavities is 3 mm; and the length L of each of the cavities is 40 mm.

[0084] In an embodiment of the third inventive aspect, the plurality of cavities comprises between 1 and 10 cavities, preferably between 1 and 5 trenches, more preferably 2 trenches.

[0085] Advantageously, in a particular embodiment of the third inventive aspect, the optimal number of trenches is two. This allows for optimal device sizing and ensures the correct extraction of platelet-rich plasma from the blood sample. In other words, the device is designed to be compact but with enough trenches to trap all the blood cells in the sample, allowing only plasma to be extracted through the device's outlet. While more trenches could be included, this would not improve plasma extraction but would simply add dead space within the microfluidic device. Conversely, fewer than two trenches could compromise the purity of the extracted plasma, potentially resulting in the inclusion of some red blood cells.

[0086] In an embodiment according to any embodiment of the foregoing inventive aspects, the hydrophilic coating is a pressure-sensitive hydrophilic coating.

[0087] Advantageously, the pressure-sensitive hydrophilic coating improves the detection of the blood sample entering the channel, particularly within the plurality of cavities, and prevents the formation of bubbles within the device once it is filled with the blood sample.

[0088] In an embodiment according to any embodiment of the above inventive aspects, the depth C of the microchannel is 300 pm.

[0089] Advantageously, a microchannel with a depth of 300 pm allows the addition of red blood cells more easily and also provides a smaller dead volume within the device of the invention, i.e., it allows the device to be sized in a more compact way.

[0090] In an embodiment according to any embodiment of the foregoing inventive aspects, the channel comprises at least two straight and parallel sections, the at least two sections being fluidly connected.

[0091] By comprising at least two straight and parallel sections, it allows the manufacture of a more compact device.

[0092] In one embodiment, at least one of the at least two straight sections comprises the plurality of cavities. In a particular embodiment, the first section is cavity-free and the second section comprises the plurality of cavities.

[0093] In one particular embodiment, the at least two straight sections are joined by means of an additional U-shaped section.

[0094] Advantageously, the shape of II of the additional section joining the at least two straight frames allows for an even more compact shape to be given to the device of the invention by joining the at least two sections smoothly.

[0095] In an embodiment according to any of the foregoing inventive aspects, the channel comprises at least a first portion configured to store a blood sample and disposed after the inlet, and at least a second portion, disposed after the at least a first portion, comprising the microchannel and the plurality of cavities, the at least a second portion being configured to separate the plasma from the blood and being fluidly connected to the at least a first portion.

[0096] The term “a continuation de,” as used in this description, refers to a portion or element being immediately after a referenced portion or element in the flow direction, that is, from the inlet to the outlet of the microfluidic device channel. Thus, in this embodiment, at least one first portion is immediately after the channel inlet, i.e., a continuation of the channel inlet. Likewise, at least one second portion is immediately after the at least one first portion, i.e., a continuation of the at least one first portion.

[0097] At least the first portion of the channel allows sedimentation of the blood cells to begin before the blood sample enters the second portion, that is, the portion comprising the microchannel and the plurality of cavities. Therefore, some of the blood cells in the sample remain trapped within this first portion, since the first cavity of the second portion also acts as the first plasma separator.

[0098] In a fourth inventive aspect, the invention provides a method for obtaining platelet-enriched plasma from a blood sample comprising the following steps: a) providing a microfluidic device according to any of the preceding inventive aspects, b) introducing a predetermined volume of blood sample through the channel inlet, c) extracting a volume of plasma by means of a pump fluidly connected to the channel outlet.

[0099] All the definitions described above in relation to the first inventive aspect are equally applicable to the fourth inventive aspect and its particular embodiments.

[0100] Any embodiment of the device of the invention allows for the processing of blood samples on the order of milliliters thanks to their respective specific dimensions and the plurality of cavities. That is to say, in one embodiment of the method, in step b), a predetermined volume of at least 1 mL is introduced. Preferably, the predetermined blood sample volume is 1 mL.

[0101] In one embodiment, the process of obtaining platelet-rich plasma from the blood sample ends when it is detected that some blood cells reach the outlet of the device. In this case, it is when the plurality of cavities, and more generally the microfluidic device, is no longer capable of separating more blood cells from the plasma.

[0102] In an embodiment of the fourth inventive aspect of the invention, the blood sample introduced into the microfluidic device during step b) has a temperature between 2 and 37°C.

[0103] In a particular embodiment of the fourth inventive aspect of the invention, the blood sample introduced into the microfluidic device during step b) has a temperature between

[0104] 3 and 36°C, between 4 and 35°C, between 5 and 34°C, between 6 and 33°C, between 7 and 32°C, between

[0105] 8 and 31°C, between 9 and 30°C, between 10 and 29°C, between 11 and 28°C, between 12 and 27°C, between 13 and 26°C, between 14 and 25°C, between 15 and 24°C, between 16 and 23°C, between 17 and 22°C, between 18 and 21°C, or between 19 and 20°C. In another particular embodiment of the fourth inventive aspect of the invention, the blood sample introduced into the microfluidic device during step b) has a temperature between 2 and 35°C, between 2 and 33°C, between 2 and

[0106] 31°C, between 2 and 29°C, between 2 and 27°C, between 2 and 25°C, between 2 and 23°C, between 2 and

[0107] 21°C, between 2 and 19°C, between 2 and 17°C, between 2 and 15°C, between 2 and 13°C, between 2 and

[0108] 11°C, between 2 and 9°C, between 2 and 7°C, between 2 and 5°C, or between 2 and 3°C. In another particular embodiment of the fourth inventive aspect of the invention, the blood sample introduced into the microfluidic device during step b) has a temperature between

[0109] 4 and 37°C, between 6 and 37°C, between 8 and 37°C, between 10 and 37°C, between 12 and 37°C, between 14 and 37°C, between 16 and 37°C, between 18 and 37°C, between 20 and 37°C, between 22 and 37°C, between 24 and 37°C, between 26 and 37°C, between 28 and 37°C, between 30 and 37°C, between

[0110] 32 and 37°C, between 34 and 37°C, or between 36 and 37°C.

[0111] In an embodiment of the fourth inventive aspect of the invention, where the predetermined blood sample volume, the introduction of the predetermined blood sample volume in step b) and the extraction of platelet-enriched plasma in step c) are carried out in such a way as to achieve a flow rate of between 5 and 50 pL.min' 1 , preferably at a flow rate of 12.5 pL.min' 1 .

[0112] In a particular embodiment of the fourth inventive aspect of the invention, the flow rate is between 6 and 49 pL.min' 1 , between 7 and 48 pL.min' 1 , between 8 and 47 pL.min' 1 , between 9 and 46 pL.min' 1 , between 10 and 45 pL.min' 1 , between 11 and 44 pL.min' 1 , between 12 and 43 pL.min' 1 , between 13 and 42 pL.min' 1 , between 14 and 41 pL.min' 1 , between 15 and 40 pL.min' 1 , between 16 and 39 pL.min -1, between 17 and 38 pL.min -1 , between 18 and 37 pL.min -1 , between 19 and 36 pL.min -1 , between 20 and 35 pL.min -1 , between 21 and 34 pL.min -1 , between 22 and 33 pL.min -1 , between 23 and 32 pL.min -1 , between 24 and 31 pL.min -1 , between 25 and 30 pL.min -1 , between 26 and 29 pL.min -1 , or between 27 and 28 pL.min -1 In another particular embodiment of the fourth inventive aspect of the invention, the flow rate is between 5 and 48 pL.min -1 , between 5 and 46 pL.min -1 , between 5 and 44 pL.min -1 , between 5 and 42 pL.min -1 , between 5 and 40 pL.min -1 , between 5 and 38 pL.min -1 , between 5 and 36 pL.min -1 , between 5 and 34 pL.min -1 , between 5 and 32 pL.min -1 , between 5 and 30 pL.min- 1 , between 5 and 28 pL.min -1 , between 5 and 26 pL.min -1 , between 5 and 24 pL.min-1 , between 5 and 22 pL.min -1 , between 5 and 20 pL.min -1 , between 5 and 18 pL.min -1 , between 5 and 16 pL.min -1 , between 5 and 14 pL.min -1 , between 5 and 12 pL.min -1 , between 5 and 10 pL.min -1 , between 5 and 8 pL.min -1 , or between 5 and 6 pL.min -1 In another particular embodiment of the fourth inventive aspect of the invention, the flow rate is between 7 and 50 pL.min -1 , between 9 and 50 pL.min -1 , between 11 and 50 pL.min -1 , between 13 and 50 pL.min -1 , between 15 and 50 pL.min -1 , between 17 and 50 pL.min- 1 , between 19 and 50 pL.min -1 , between 21 and 50 pL.min -1 , between 23 and 50 pL.min -1 , between 25 and 50 pL.min -1 , between 27 and 50 pL.min -1 , between 29 and 50 pL.min -1 , between 31 and 50 pL.min -1 , between 33 and 50 pL.min -1, between 35 and 50 pL.min -1 , between 37 and 50 pL.min -1 , between 39 and 50 pL.min -1 , between 41 and 50 pL.min -1 , between 43 and 50 pL.min -1 , between 45 and 50 pL.min -1 , between 47 and 50 pL.min -1 , or between 49 and 50 pL.min -1 .

[0113] Advantageously, the flow rate is continuous throughout the process of obtaining platelet-rich plasma from the blood sample, which ensures that the process is continuous and that the sedimentation phenomenon occurs optimally and that the globules descend to the bottom of the channel.

[0114] Furthermore, the separation method allows for the almost immediate extraction of platelet-enriched plasma from the blood sample; that is, it does not require any waiting time for sedimentation to begin and for the cavities to act by trapping the globules. As soon as the microfluidic device is filled with a blood sample and, in operational mode, the pump coupled to the outlet of the channel is started, the plasma is extracted from the blood sample.

[0115] However, the flow rate range is between 5 and 50 pL.min -1The process is considered relatively slow, and a certain amount of time is needed for the microfluidic device channel to fill completely, from the inlet to the outlet. Specifically, throughout the process, there is a correlation between the plasma collection time and the desired plasma volume extracted from the device. The plasma volume and the collection time also depend on the shape of the multiple cavities.

[0116] Specifically, for separation times exceeding 45 minutes, elongated cavities in the flow direction allow for the extraction of a larger volume compared to any other cavity shape. However, between 20 and 40 minutes, the device with circular cavities allows for the extraction of the largest plasma volume, while the device with elongated cavities in the flow direction allows for the extraction of the least volume in the same timeframe.

[0117] In particular, there is also an increase in plasma collected that is inversely proportional to the flow rate introduced into the device. Specifically, a lower flow rate allows for better sedimentation of the globules within the device, and particularly within the plurality of cavities, which increases the amount of platelet-rich plasma extracted from the device of the invention.

[0118] In an embodiment of the fourth inventive aspect of the invention, the volume of plasma extracted from the microfluidic device is up to 300 pL per milliliter of blood sample introduced into the microfluidic device of the invention.

[0119] In a fifth inventive aspect, the invention provides the use of the microfluidic device of the invention for obtaining platelet-enriched plasma from a blood sample.

[0120] All the definitions described above in relation to the first inventive aspect are equally applicable to the fifth inventive aspect and its particular embodiments.

[0121] DESCRIPTION OF THE DRAWINGS

[0122] These and other features and advantages of the invention will become clearer from the following detailed description of a preferred embodiment, given only as an illustrative and non-limiting example, with reference to the accompanying figures.

[0123] Figure 1 This figure shows a cross-sectional view of a microfluidic device according to an example of the embodiment of the present invention.

[0124] Figure 2 This figure shows a perspective view of a microfluidic device according to an example of realization of the first inventive aspect.

[0125] Figure 3 This figure shows a perspective view of a microfluidic device according to an example of realization of the second inventive aspect.

[0126] Figure 4 This figure shows a perspective view of a microfluidic device according to an example of realization of the third inventive aspect.

[0127] Figure 5 This figure shows a perspective view of a microfluidic device according to an example of the realization of the present invention.

[0128] Figure 6 This figure shows a cross-sectional view of a microfluidic device according to an example of the embodiment of the present invention, showing the sedimentation process that occurs along the device.

[0129] Figure 7 This figure shows an image of an individual cavity of a microfluidic device according to an example of embodiment of the present invention showing the filling differences using a regular pressure-sensitive coating (PSA) or a hydrophilic pressure-sensitive coating (PSA) at the bottom of the cavity.

[0130] Figure 8 This figure shows the plasma collection volume from the three device designs (device with rectangular trenches (black bars), device with circular trenches (gray bars), and device with two large trenches (white bars)) (Figure 8A), the progression of the volume over time of the whole blood meniscus (line with hexagons) and the GR meniscus (line with diamonds) during a separation experiment according to an embodiment of the present invention (Figure 8B), and the progression of the plasma generation volume over time during an experiment using the three device designs at flow rates of 12.5 pL min -1 (Figure 8C) and 25.0 pL min -1 (Figure 8D). All graphs show mean and standard deviation values. N = 3.

[0131] Figure 9. This figure shows the plasma composition results obtained using a hematology analyzer on plasma samples collected from a microfluidic device, according to an example of the embodiment of the present invention, after processing the same whole blood sample (Figure 9A) or after processing three different whole blood samples (Figure 9B) for white blood cells (WBCs), red blood cells (RBCs), and platelets (PLTs). The device with rectangular trenches is represented by black bars, the device with circular trenches by gray bars, and the device with two large trenches by white bars. Mean and standard deviation above baseline values ​​(% of baseline whole blood concentrations), N = 3.

[0132] Figure 10. This figure shows the results of the platelet activation assay. Figure 10A shows representative flow cytometry results, including total platelets (D2 and D4) and activated platelets (D2). Figure 10B shows the platelet activation results for the three device designs (rectangular trench device (RT), circular trench device (CT), and two large trench device (TbT)) along with the conventional centrifugation (CTF) method.

[0133] DETAILED EXPLANATION OF THE INVENTION

[0134] The present invention, according to the first, second and third inventive aspects, is a microfluidic device (1) for obtaining platelet-enriched plasma from a blood sample.

[0135] Figure 1 shows a representation of the microfluidic device (1) for separating plasma from a blood sample. The microfluidic device (1) comprises a channel (2) with an inlet (3), an outlet (4), an upper section (5), and a lower section (6). A height H is defined between the upper section (5) and the lower section (6), representing the height of the channel (2) and ranging from 1 to 3 mm. The inlet (3) of the channel (2) is configured for introducing a blood sample, and the outlet (4) of the channel (2) is configured to fluidly couple a pump (7), which is configured to extract plasma from the channel (2). The channel (2) also has a length T ranging from 30 to 90 mm.

[0136] The blood sample introduced into the microfluidic device (1) through the inlet (3) comprises plasma and blood cells. The microfluidic device (1) allows the separation of the plasma from the other components of the blood sample and the extraction of said plasma through the outlet (4) of the microfluidic device (1).

[0137] In operative mode, when a blood sample is introduced through the inlet (3), there is a flow direction that is formed from the inlet (3) to the outlet (4) of the channel (2), this flow direction being represented in figure 1 by means of the arrows located inside the channel (2).

[0138] The microfluidic device (1) also comprises a plurality of cavities (8) distributed along at least a portion of the channel (2). Each of the cavities (8) comprises a lower surface (8.1) and an upper surface (8.2). Between the upper part (5) of the channel (2) and the upper surface (8.2) of the plurality of cavities (8) a microchannel (9) of depth C is formed, and where said depth C is between 100 and 400 pm.

[0139] As shown in Figure 1, there is a separation distance S between each pair of adjacent cavities (8), and this distance S is between 0.5 and 1.5 mm. Furthermore, in any embodiment of the microfluidic device (1) of the invention, each cavity (8) has a height X, which is defined by the relation X = H - C, and each cavity (8) comprises a hydrophilic coating on its lower surface (8.1). The hydrophilic coating on the lower surface (8.1) of the cavities (8) prevents the formation of bubbles within the microfluidic device (1), particularly within the cavities (8), and especially when, in operating mode, the microfluidic device (1) is empty and is filled with the blood sample.Regardless of the representation of the shape of the cavities (8) of the microfluidic device (1) in Figure 1, said cavities (8) could be either elongated in a direction perpendicular to the flow direction according to an embodiment of the first inventive aspect of the invention, or circular according to an embodiment of the second inventive aspect of the invention, or elongated in the flow direction according to an embodiment of the third inventive aspect of the invention.

[0140] Once in operating mode, the microfluidic device (1) allows the separation of plasma from the blood sample introduced through the inlet (3). The introduced blood sample comprises plasma and red blood cells. Along the channel (2) of the microfluidic device (1), these red blood cells are carried by means of the microchannel (9) and the flow created between the inlet (3) and the outlet (4), passing through successive cavities (8) of the microfluidic device (1). In each cavity (8), the red blood cells are trapped until only platelet-enriched plasma is extracted through the outlet (4) of the microfluidic device (1). In other words, the cavities (8) act as a passive and progressive filter that retains the red blood cells, in combination with the sedimentation process, to extract platelet-rich plasma free of red blood cells through the outlet (4) of the channel (2).

[0141] Furthermore, the microfluidic device (1) allows for the passive processing of blood samples on the order of milliliters based on its cavities (8) and on the sedimentation of the globules within the same cavities (8).

[0142] In one embodiment, the microfluidic device (1) comprises a plurality of layers, preferably at least one layer comprising acrylic, i.e., polymethyl methacrylate (PMMA). In a particular embodiment, the microfluidic device (1) further comprises a transparent, pressure-sensitive coating.

[0143] Figures 2 to 4 are perspective views of different embodiments of a microfluidic device (1) where an enlarged view of the plurality of cavities (8) can be observed.

[0144] Figure 2 shows a perspective view of a microfluidic device (1) according to an embodiment of the first inventive aspect, wherein each cavity (8) of the plurality of cavities (8) is elongated in a direction perpendicular to the flow direction. In any embodiment of the first inventive aspect of the microfluidic device (1), each cavity (8) comprises a height X, a width A between 0.5 and 2.5 mm, and a length L between 1.5 and 4.5 mm.

[0145] In an embodiment of the first inventive aspect of the invention, the height H of the channel (2) is 2 mm, the width A of each of the cavities (8) is 1.5 mm and the length L of each of the cavities (8) is 3 mm.

[0146] In an embodiment of the first inventive aspect of the invention, the plurality of cavities (8) comprises between 10 and 80 cavities, preferably between 25 and 65 trenches, more preferably 45 trenches.

[0147] Figure 3 shows a perspective view of a microfluidic device (1) according to an embodiment of the second inventive aspect, where the plurality of cavities (8) are circular. In any embodiment of the second inventive aspect of the microfluidic device (1), each of the cavities (8) comprises a height X and a diameter D between 1.5 and 4.5 mm.

[0148] In an embodiment of the second inventive aspect, the height H of the channel (2) is 2 mm; and the diameter D of each of the cavities (8) is 3 mm.

[0149] In an embodiment of the second inventive aspect, the plurality of cavities (8) comprises between 10 and 60 cavities, preferably between 20 and 45 trenches, more preferably 30 trenches.

[0150] Figure 4 shows a perspective view of a microfluidic device (1) according to an example of the third inventive aspect and where each cavity (8) of the plurality of cavities (8) has an elongated shape in the flow direction.

[0151] In one embodiment of the third inventive aspect, the height of the channel (2) is 2 mm; the width A of each of the cavities (8) is 3 mm; and the length L of each of the cavities (8) is 40 mm. In one embodiment of the third inventive aspect, the plurality of cavities (8) comprises between 1 and 10 cavities, preferably between 1 and 5 trenches, more preferably 2 trenches.

[0152] In an embodiment of any of the inventive aspects of the invention, the hydrophilic coating is a pressure-sensitive hydrophilic coating.

[0153] In an embodiment of any of the inventive aspects of the invention, the depth C of the microchannel (9) is 300 pm.

[0154] Figure 5 shows a perspective view of a microfluidic device (1) according to an embodiment of the third inventive aspect of the invention. In Figure 5, the shape of the cavities (8) could also be represented according to an embodiment of the first inventive aspect of the invention or according to an embodiment of the second inventive aspect.

[0155] In an embodiment of any of the inventive aspects of the invention, the channel (2) comprises at least two straight (2.1, 2.2, 2.3, 2.4) sections parallel to each other, the at least two sections (2.1, 2.2, 2.3, 2.4) being fluidly joined.

[0156] In an embodiment of any of the inventive aspects of the invention, the channel (2) comprises at least a first portion (2.6) configured to store a blood sample and at least a second portion (2.7) comprising the microchannel (9) and the plurality of cavities (8), the at least a second portion (2.5) being configured to separate the plasma from the blood and being fluidly connected to the at least a first portion (2.4).

[0157] In the example in Figure 5, the channel (2) comprises four straight sections (2.1, 2.2, 2.3, 2.4) fluidly connected to each other by means of additional sections (2.5) in the shape of an II. A first additional section (2.5) connects the first straight section (2.1) to the second straight section (2.2). A second additional section (2.5) connects the second straight section (2.2) to the third straight section.

[0158] (2.3). A third additional section (2.5) joins the third straight section (2.3) with the fourth straight section

[0159] (2.4).

[0160] Furthermore, the microfluidic device (1) of Figure 5 comprises a first portion (2.6) and subsequently a second portion (2.7), both portions being fluidically connected. The first portion (2.6), represented by a discontinuous rectangle near the inlet (3) of the microfluidic device (1), comprises the first straight section (2.1) and the second straight section (2.2). The blood sample is introduced into the first portion (2.6) before being directed to the plurality of cavities (8). The second portion (2.7), represented by a discontinuous rectangle near the outlet (4) of the microfluidic device (1), comprises the third straight section (2.3) and the fourth straight section (2.4). The first portion (2.6) is in fluid communication with the second portion (2.7).7) and allows storage of the blood sample before entering the microchannel and the plurality of cavities (8), which allows the separation of the plasma from the blood sample and the obtaining of a high purity plasma enriched with platelets.

[0161] Figure 6 shows a cross-sectional view of the device in Figure 1, where it can again be seen that the microfluidic device (1) comprises a channel (2) with an inlet (3), an outlet (4), an upper part (5), and a lower part (6). The inlet (3) of the channel (2) is configured for the introduction of a blood sample, and the outlet (4) of the channel (2) is configured to fluidly couple a pump (7). This pump (7) is configured to extract plasma from the channel (2). Figure 6 is particularly interesting for observing the sedimentation process of blood cells that occurs along the channel (2) of the microfluidic device (1) to obtain high-purity, platelet-rich plasma at the outlet (3).

[0162] Figures 7 to 10 describe the results of an experimental example according to the microfluidic device of the invention.

[0163] Figure 7 shows an image of an individual cavity of the device in Figure 3 (device with circular trenches), illustrating the filling differences using either a regular pressure-sensitive adhesive (PSA) coating or a hydrophilic pressure-sensitive adhesive (PSA) coating at the bottom of the cavity. Red food coloring in distilled water was used to measure bubble formation, and images were taken during device filling at a flow rate of 50.0 pL / min. -1 .

[0164] Figure 7 shows that when the regular pressure-sensitive coating is used, the liquid moves through the cavity walls and the top layer because they are in contact with the flowing liquid. Eventually, the liquid reaches the bottom, but the hydrophobicity of the regular pressure-sensitive coating prevents homogeneous and reproducible filling in each cavity. This uneven filling leads to bubble formation in several cavities of the device. On the other hand, when using the hydrophilic pressure-sensitive coating, the liquid can easily wet and flow across the entire bottom of the cavity, allowing for more controlled filling from the bottom up. The same filling behavior is observed when using the devices in Figures 2 and 4 (device with rectangular trenches and device with two large trenches, respectively).Furthermore, by using the hydrophilic pressure-sensitive coating, the filling of each cavity is reproducible and reliable.

[0165] Figure 8A shows the plasma volume collected from the three device designs (devices with rectangular trenches, circular trenches, or two large trenches, corresponding to the devices in Figures 2, 3, and 4, respectively). For each plasma separation assay, 1 mL of whole blood was pipetted into the device before connecting a syringe pump to the device's channel outlet. The three device designs were tested at three flow rates (12.5, 25.0, 37.5, and 50.0 pL / min). -1 To measure the amount of plasma collected in a syringe after each experiment. An increase in plasma collected was observed that was inversely proportional to the flow rate, with a maximum collected volume of 300 pL for a flow rate of 12.5 pL / min -1However, experiments at low flow rates were slower, for example, at a flow rate of 12.5 pL min -1 It required 65 min versus 30 min and 15 min for flow rates of 25.0 and 37.5 pL min -1 , respectively, while 50.0 pL min- 1No plasma separation was observed. The test time is likely the main reason for the different plasma collected, as at low flow rates, the blood has more time to settle within the plasma separator. Furthermore, significant differences in the amount of plasma collected were observed between the device designs at the same flow rates. For example, while the devices with rectangular and circular trenches (devices in Figures 2 and 3, respectively) showed no significant differences in plasma collected, the device with two large trenches (device in Figure 4) collected approximately 11% and 6% more plasma than the others at flow rates of 25.0 and 12.5 pL / min. -1respectively. This difference in the collected volume is probably due to an agitation effect caused by repetitive abrupt changes in the fluid cross-section in the devices with rectangular and circular trenches in Figures 2 and 3, which is not present in the device with two large trenches in Figure 4. Overall, it is shown that the three device designs can separate whole blood plasma at different flow rates with high reproducibility.

[0166] Figure 8B shows the progression of whole blood volume (hexagon line) and red blood cells (diamond line) over time in a rectangular trench device (device from Example 2). As expected, the whole blood flow (hexagon line) increased linearly with time; a linear trend line (R² = 0.9997) was generated with a slope of 12.54, corresponding to a flow rate of 12.5 pL / min.-1 used in the syringe pump. This confirms the desired blood flow rate through the plasma separator and its stability during the experiments. On the other hand, the progress of the red blood cell (RBC) meniscus (diamond line) is affected by sedimentation and trenches in the plasma separator, with a decrease in the RBC flow rate observed after the first 10 minutes of this experiment. However, the flow rate recovers to 80% of its velocity after 30 minutes of the experiment. Subsequently, the RBC flow rate eventually reaches the same velocity as the whole blood, indicating that plasma separation is complete. In fact, the difference between the whole blood and RBC flow rates results in the plasma generated at a given time. Therefore, the dynamic plasma generation over time can be calculated for each device design at different flow rates (Figures 8C-D).

[0167] For experiments using a flow rate of 12.5 pL min -1 (Figure 8C), a similar plasma generation dynamic was observed between the devices with rectangular and circular trenches (devices in Examples 2 and 3, respectively), while the device with two large trenches (device in Example 4) showed a 10-minute delay in initiating plasma generation. The experiments at 25.0 pL min -1 (Figure 8D) showed no significant differences between the different trench devices. Surprisingly, for the 12.5 and 25.0 pL min experiments -1 Although there is a difference in flow rates, they can generate the same amount of plasma at the same time. For example, after 30 minutes, both have approximately 200 pL of plasma already separated. This supports the idea that plasma generation is primarily due to sedimentation time within the device and not to flow rate.

[0168] Figure 9 shows the plasma composition results obtained from plasma samples collected from the three device designs (devices with rectangular trenches, circular trenches, or two large trenches, corresponding to the devices in Figures 2, 3, and 4, respectively) analyzed using a hematology analyzer. Figure 9A shows the concentrations above baseline (% of baseline whole blood concentrations) of WBCs, RBCs, and platelets for the three device designs using whole blood from the same subject. The assay reproducibility is shown with a coefficient of variation (CV) of 32%, 24%, and 8% for WBCs, RBCs, and platelets, respectively. RBC purity, in terms of elimination, was up to 98%. Surprisingly, platelets were concentrated 2.5-fold for all three device designs, likely due to the elimination of the RBC volume.In experiments using samples from three different subjects (Figure 9B), CVs of 74%, 25%, and 21% were observed for WBCs, RBCs, and platelets, respectively, which are higher likely due to the significant differences between the blood samples of each subject. However, a purity of 98% and a platelet concentration twice as high were still observed for all three devices. No significant differences in plasma components were observed for the devices with rectangular or circular trenches (devices in Figures 2 and 3, respectively); however, the device with two large trenches (device in Figure 4) showed a reduced WBC concentration compared to the others.Possibly, the long cavities in the device with two large trenches allow the sedimentation of GB along with GR, while devices with rectangular or circular trenches with smaller cavities keep GB afloat due to abrupt changes in flow rates.

[0169] Figure 10 shows the results of the platelet activation assay using the three device designs (rectangular trench devices, circular trench devices, or two large trench devices, corresponding to the devices in Figures 2, 3, and 4, respectively) with three different whole blood samples. Platelet activation was measured in samples separated by a conventional centrifugation method for comparison. Phycoerythrin-labeled anti-CD62p antibodies (561921, BD, USA) and fluorescein isothiocyanate-labeled anti-CD41A antibodies (561851, BD, USA) were used as platelet activation markers for the platelet activation assay. The plasma obtained from the experiments was prepared as follows: first, an antibody cocktail was prepared of 5 pL of anti-CD62p and 5 pL of anti-CD41A diluted in 80 pL of PBS (Gibco pH 7.4, Fisher Scientific, USA).) for each sample analysis. Subsequently, 10 pL of plasma samples were mixed with the antibody solution and incubated for 15 minutes in the dark. The platelets were then fixed with a 1.25% formaldehyde solution in PBS prepared from a 4% formaldehyde reagent (15423179, Fisher Scientific, ULI, USA). Finally, a ten-color flow cytometer (Gallios, Beckman Coulter, IE) was used for sample analysis. The data were analyzed using Gallios 1.2 software.

[0170] According to the results obtained in Figure 10, the separation devices of the invention appear to damage platelets much less than the conventional centrifugation method. The activation levels of the two methods were compared by measuring the expression levels of P-selectin (CD62p), a commonly used indicator of platelet activation. Samples were measured by flow cytometry, labeling platelets with anti-CD62p antibodies conjugated to PE and anti-CD41 antibodies conjugated to FITC as a positive control to measure all platelets. Figure 10A shows a representative result of platelet fluorescence levels for one of the devices, where it can be seen that only 8.2% of the total platelets are activated (D2).Figure 10B summarizes the platelet activation results for the three device designs of the invention (device with rectangular trenches (RT), device with circular trenches (CT), and device with two large trenches (TbT)) and the conventional centrifugation method (CTF). All three device designs of the invention showed less platelet activation than the conventional centrifugation method. This demonstrates that platelets experience less stress within these devices compared to centrifugation. Furthermore, centrifugation can cause irreversible changes in platelet morphology and function, limiting its future applications.

[0171] The present invention, according to the fourth inventive aspect of the invention, is a method for obtaining platelet-enriched plasma from a blood sample.

[0172] The method for obtaining platelet-enriched plasma from a blood sample comprises the following steps: a) providing a microfluidic device (1) according to any of the claims of any of the first three inventive aspects of the invention, b) introducing a predetermined volume of blood sample through the inlet (3) of the channel (2), c) extracting a volume of plasma by means of a pump (7) fluidly connected to the outlet (4) of the channel (2).

[0173] In one embodiment of the fourth inventive aspect, the blood sample introduced into the microfluidic device (1) during step b) has a temperature between 2 and 37°C. In one embodiment of the fourth inventive aspect, the predetermined volume of blood sample is introduced, in step b), at a flow rate between 5 and 50 pL / min. 1 , preferably at a flow rate of 12.5 pL.min' 1 .

Claims

37 CLAIMS 1. A microfluidic device (1) for obtaining platelet-enriched plasma from a blood sample, wherein the microfluidic device (1) comprises: - a channel (2) comprising an inlet (3) and an outlet (4), an upper part (5) and a lower part (6), and a height H defined between the upper part (5) and the lower part (6) of the channel (2), the inlet (3) being configured for the introduction of a blood sample, the outlet (4) being configured to fluidly couple a pump (7) configured to extract plasma from the channel (2), and comprising a flow direction from its inlet (3) to its outlet (4); - a plurality of cavities (8) distributed along at least a portion of the channel (2), each of the cavities (8) comprising a lower surface (8.1) and an upper surface (8.2), and where a microchannel (9) is formed between the upper part (5) of the channel (2) and the upper surface (8.2) of the plurality of cavities (8); where each cavity (8) of the plurality of cavities (8) is elongated in a direction perpendicular to the flow direction, and where each cavity (8) comprises a height X; a width A, said width A being between 0.5 and 2.5 mm; a length L, said length L being between 1.5 and 4.5 mm; between any two adjacent cavities (8) there is a separation distance S between them, said distance S being between 0.5 and 1.5 mm; the channel (2) comprises a height H being between 1 and 3 mm; and a length T, said length T being between 30 and 90 mm; and the microchannel (9) comprises a depth C, said depth C being between 100 and 400 mm; and. 38 wherein each of the cavities (8) comprises a hydrophilic coating on its lower surface (8.1) and the height X of each of the cavities (8) is defined by the relaton X = H - C. 2.- Microfluidic device (1) according to claim 1, where the height H of the channel (2) is 2 mm; the width A of each of the cavities (8) is 1.5 mm; and the length L of each of the cavities (8) is 3 mm.

3. Microfluidic device (1) for obtaining platelet-enriched plasma from a blood sample, wherein the microfluidic device (1) comprises: - a channel (2) comprising an inlet (3) and an outlet (4), an upper part (5) and a lower part (6), and a height H defined between the upper part (5) and the lower part (6) of the channel (2), the inlet (3) being configured for the introduction of a blood sample, the outlet (4) being configured to fluidly couple a pump (7) configured to extract plasma from the channel, and comprising a flow direction from its inlet (3) to its outlet (4); - a plurality of cavities (8) distributed along at least a portion of the channel (2), each of the cavities (8) comprising a lower surface (8.1) and an upper surface (8.2), and wherein a microchannel (9) is formed between the upper part (5) of the channel (2) and the upper surface (8.2) of the plurality of cavities (8); wherein each cavity (8) of the plurality of cavities (8) is circular in shape, and wherein each cavity (8) comprises a height X; a diameter D, said diameter D being between 1.5 and 4.5 mm; between any two adjacent cavities (8) a separation distance S, said distance S being between 0.5 and 1.5 mm; The channel (2) comprises a height H between 1 and 3 mm; and a length T, said length T being between 30 and 90 mm; and the microchannel (9) comprises a depth C, said depth C being between 100 and 400 mm; and wherein each of the cavities (8) comprises a hydrophilic coating on its lower surface (8.1) and the height X of each of the cavities (8) is defined by the relation X = H - C. 4.- Microfluidic device (1) according to claim 3, wherein the height H of the channel (2) is 2 mm; and the diameter D of each of the cavities (8) is 3 mm.

5. Microfluidic device (1) for obtaining platelet-enriched plasma from a blood sample, wherein the microfluidic device (1) comprises: - a channel (2) comprising an inlet (3) and an outlet (4), an upper part (5) and a lower part (6), and a height H defined between the upper part (5) and the lower part (6) of the channel (2), the inlet (3) being configured for the introduction of a blood sample, the outlet (4) being configured to fluidly couple a pump (7) configured to extract plasma from the channel, and comprising a flow direction from its inlet (3) to its outlet (4); - a plurality of cavities (8) distributed along at least a portion of the channel (2), each of the cavities (8) comprising a lower surface (8.1) and an upper surface (8.2), and wherein a microchannel (9) is formed between the upper part (5) of the channel (2) and the upper surface (8.2) of the plurality of cavities (8); wherein each cavity (8) of the plurality of cavities (8) is elongated in the direction of flow, where Each cavity (8) comprises a height X; a width A, said width A being between 0.5 and 4.5 mm; a length L, said length L being between 20 and 60 mm; the channel (2) comprises a height H being between 1 and 3 mm; and a length T, said length T being between 30 and 90 mm; the microchannel (9) comprises a depth C, said depth C being between 100 and 400 mm; and wherein each of the cavities (8) comprises a hydrophilic coating on its lower surface and the height X of each of the cavities (8) is defined by the relation X = H - C. 6.- Microfluidic device (1) according to claim 5, where the height H of the channel (2) is 2 mm; the width A of each of the cavities (8) is 3 mm; and the length L of each of the cavities (8) is 40 mm. 7.- Microfluidic device (1) according to any of the preceding claims, wherein the hydrophilic coating is a pressure-sensitive hydrophilic coating. 8.- Microfluidic device (1) according to any of the preceding claims, wherein the hydrophilic coating comprises a material selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polydopamine and polyacrylic acid. 9.- Microfluidic device (1) according to any of the preceding claims, wherein the depth C of the microchannel (9) is 300 pm. 10.- Microfluidic device (1) according to any of the preceding claims, wherein the channel (2) comprises at least two straight (2.1, 2.2, 2.3, 2.4) sections parallel to each other, the at least two sections (2.1, 2.2, 2.3, 2.4) being fluidly connected. 11.- Microfluidic device (1) according to claim 10, wherein the at least two straight sections (2.1, 2.2, 2.3, 2.4) are joined by means of an additional section (2.5) in the shape of II. 12.- Microfluidic device (1) according to any of the preceding claims, wherein the channel (2) comprises at least a first portion (2.6) configured to store the blood sample and disposed after the inlet (3), and at least a second portion (2.7), disposed after the at least a first portion (2.6), comprising the microchannel (9) and the plurality of cavities (8), the at least a second portion (2.7) being configured to separate the plasma from the blood and being fluidly connected to the at least a first portion (2.6). 13.- Method for obtaining platelet-enriched plasma from a blood sample comprising the following steps: a) providing a microfluidic device (1) according to any of the preceding claims, b) introducing a predetermined volume of blood sample through the inlet (3) of the channel (2), c) extracting a volume of plasma by means of a pump (7) fluidically connected to the outlet (4) of the channel (2).

14. Method according to claim 13, wherein the blood sample introduced into the microfluidic device (1) during step b) has a temperature between 2 and 37°C.

15. Method according to any of claims 13 or 14, wherein the predetermined blood sample volume, the introduction of the predetermined blood volume in step b) and the extraction of the platelet-enriched plasma in step c) are carried out in such a way as to achieve a flow rate of between 5 and 50 pL.min' 1, preferably at a flow rate of 12.5 pL.min' 1 . 16.- The microfluidic device (1) according to any of claims 1 to 12 for obtaining platelet-enriched plasma from a blood sample.

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