Method for obtaining a blood-derived product and blood-derived product thus obtained

Abstract

The current invention relates to a method for obtaining a blood-derived product, wherein said blood-derived product is chosen from an α2-macroglobulin enriched sample The current invention further relates to a blood-derived product obtained by aforementioned method and use of said blood-derived product in the treatment of a musculoskeletal disease or a respiratory disease. The current invention further 10 relates to a concentrator tube and kit for obtaining an α2-macroglobulin enriched sample.

Description

[0001] METHOD FOR OBTAINING A BLOOD-DERIVED PRODUCT AND BLOOD-

[0002] DERIVED PRODUCT THUS OBTAINED

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a method for obtaining a blood-derived product, wherein optionally an incubation step is performed on a whole blood sample or on a fractionated blood sample. In further aspects, the present invention also relates to a blood-derived product thus obtained and use of said blood-derived product in the treatment of a musculoskeletal disease, preferably a bone disease, a tendon or ligament disease or a joint disease in a subject. The invention further relates to use of said blood-derived product in the treatment of a respiratory disease and to a concentrator tube and kit for obtaining such a blood-derived product.

[0005] BACKGROUND

[0006] Platelet-rich plasma (PRP) has been clinically used as an easily prepared growth factor cocktail that can promote wound healing, angiogenesis, and tissue remodeling. It was revealed through a number of studies that various bioactive substances, including platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-p), and epidermal growth factor (EGF), were discharged from the a-granules of platelets into plasma when the platelets were destroyed and activated.

[0007] Despite the increasing use of PRP therapeutically, its reported clinical effects are quite variable. And although many commercialized devices are now available for the clinical preparation of PRP, there is no standardized protocol for PRP preparation that efficiently maximizes platelet yield, platelet activation, while decreasing platelet aggregation. The current protocols often rely on calcium activation to increase the release of growth factors from the platelets. However, CaCH addition complicates handling, particularly in view of the tightening regulations for blood-derived products.

[0008] Furthermore, there remains a need in the art for an improved method for obtaining blood-derived products having an enrichment of certain beneficial proteins, such as blood-derived products having an enrichment of alpha 2 macroglobulin (02- Macroglobulin, A2M or A2MG) or Interleukin Receptor Antagonist Protein (IRAP). Insulin-like growth factors (IGFs) are crucial for many aspects of development, growth, and metabolism. A2MG is a binding protein of Insulin-like growth factorbinding protein 1 (IGFBP-1) resulting in enhanced IGF effects. Interleukin Receptor Antagonist Protein (IRAP) prevents IL-1 from binding to cells within the joint and exerting its degradative effects.

[0009] The aim of the invention is to provide a method which eliminates those disadvantages.

[0010] The invention thereto aims to provide an optimized method for preparing blood- derived products such as PRP and samples enriched for certain beneficial proteins.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention and embodiments thereof serve to provide a solution to one or more of above-mentioned disadvantages.

[0013] To this end, the present invention relates to a method for obtaining a blood-derived product according to claim 1.

[0014] The current invention provides a reliable and practical method to maximize the yield of qualitatively optimized blood-derived products using common laboratory ware and equipment. Preferred embodiments of the method are shown in any of the claims 2 to 13.

[0015] In a second aspect, the present invention relates to a blood-derived product according to claim 14. Preferred embodiments of the blood-derived product are shown in any of the claims 15 to 18.

[0016] In a further aspect, the invention relates to use of aforementioned blood-derived products for use in the treatment of a musculoskeletal disease, preferably a bone disease, a tendon disease or a joint disease in a subject.

[0017] In a further aspect, the invention relates to aforementioned blood-derived products for use in the treatment of a respiratory disease.

[0018] In a further aspect, the invention relates to a concentrator tube for obtaining an a2- macroglobulin enriched sample. In a last aspect, the invention relates to a kit comprising aforementioned concentrator tube.

[0019] DESCRIPTION OF FIGURES

[0020] Figure 1 shows a2-macroglobulin protein levels (1A-B), platelets levels (1C) and IGF-1 protein levels (1D-E) as obtained by various blood processing methods, including the method of the current invention for obtaining an a2-macroglobulin enriched sample and a PRP sample.

[0021] Figure 2 shows serum IRAP levels (ng / ml) in blood-derived samples obtained by various methods, including the method according to an embodiment of the present invention ("24h Cytolease").

[0022] Figure 3 shows serum levels (ng / ml) of IL-1, IL-10 and IRAP in blood-derived samples obtained by various methods having an incubation step of 24h at 37°C, including the method according to an embodiment of the present invention ("Cytolease").

[0023] Figure 4 shows a concentrator tube with a concentrator column having a molecular weight cut-off filter membrane with a conical shape for obtaining an a2-macroglobulin enriched sample according to an embodiment of the invention.

[0024] Figure 5 shows a septum cap having multiple septa according to an embodiment of the invention.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention concerns a method for obtaining a blood-derived product, wherein said blood-derived product is chosen from platelet rich plasma (PRP), an a2- macroglobulin enriched sample or an Interleukin-1 receptor antagonist (IRAP) enriched sample. The invention further relates to said blood-derived products thus obtained and use of said blood-derived products in the treatment of a musculoskeletal disease, preferably a bone disease, a tendon or ligament disease or a joint disease in a subject. The invention also relates to aforementioned blood-derived products for use in the treatment of a respiratory disease. Furthermore, the invention also relates to a concentrator tube for obtaining an a2-macroglobulin enriched sample and a kit comprising aforementioned concentrator tube.

[0027] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.

[0028] As used herein, the following terms have the following meanings:

[0029] "A", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.

[0030] "About" as used herein referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of + / - 20% or less, preferably + / -10% or less, more preferably + / -5% or less, even more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, in so far such variations are appropriate to perform in the disclosed invention. However, it is to be understood that the value to which the modifier "about" refers is itself also specifically disclosed.

[0031] "Comprise", "comprising", and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.

[0032] The term "growth factor" as used herein refers to a biologically active substance which influences proliferation, growth, differentiation, survival and / or migration of various cell types, and may affect developmental, morphological and functional changes in an organism, either alone or when modulated by other substances. A growth factor may typically act by binding, as a ligand, to a receptor (e.g., surface or intracellular receptor) present in cells. The term "anti-coagulant" as used herein refers to a composition that can inhibit the coagulation of blood. Examples of anticoagulants used in the present invention include CPDA-1 (comprising citrate, phosphate, dextrose, and / or adenosine), heparin, citrate, oxalate, ACD-A (Acid Citrate Dextrose Solution A) or EDTA.

[0033] The term "plasma" as used herein is interchangeably with "blood plasma" and refers to the liquid component of whole blood, which makes up approximately 55% of the total blood volume. It is composed primarily of water with small amounts of minerals, salts, ions, nutrients, and proteins in solution. In whole blood, red blood cells, leukocytes, and platelets are suspended within the plasma. Plasma contains a large variety of proteins including albumin, immunoglobulins, and clotting proteins such as fibrinogen.

[0034] The term "buffy coat" as used herein refers to the fraction of non-coagulated blood, preferably obtained by means of a density gradient centrifugation, whereby the fraction is enriched with white blood cells and platelets.

[0035] The term "treatment" as used herein refers to prophylactic and / or therapeutic measures to reduce or prevent pathological conditions or disorders from developing or progressing.

[0036] The term "local administration" as used herein refers to a parenteral administration in or in the vicinity of a targeted site within the body.

[0037] The terms "patient" or "subject", are used interchangeably and refer to a mammalian subject to be treated. Preferably, the mammal is an equine, a feline or a canine.

[0038] "Feline" or "felines" in the present invention refers to cats of the Felidae family. A member of this family is also called a felid. The living Felidae are divided in two subfamilies: the Pantherinae and Felinae. Pantherinae includes five Panthera and two Neofelis species, while Felinae includes the other 34 species in ten genera, amongst which domestic cats, cheetahs, servals, lynx' and cougars.

[0039] "Canine" or "canines" in the present invention refers to dog-like carnivorans of the Canidae family. A member of this family is called a canid. There are three subfamilies found within the canid family, which are the extinct Borophaginae and Hesperocyoninae, and the extant Caninae. The Caninae are known as canines, and include domestic dogs, wolves, foxes, coyotes, jackals and other extant and extinct species.

[0040] The term "plasma" is to be understood as a fraction obtained from a sample of whole blood, provided or contacted with an anticoagulant, e.g., CPDA-1 (comprising citrate, phosphate, dextrose, and / or adenosine) heparin, citrate, oxalate, ACD-A or EDTA. Cellular components of the blood sample, i.e. white and red blood cells are separated from the liquid component, i.e. plasma, by an appropriate technique e.g., centrifugation or apheresis. In an embodiment, the plasma may include centrifuged or apheresed plasma. The mammalian plasma may be fresh frozen plasma.

[0041] Preferably, the whole blood withdrawn from a blood donor may be a mammalian whole blood sample, such that the blood-derived products are particularly suited for administration to mammalian subjects. In an embodiment, the blood is allogeneic. The collection process of allogeneic blood is well-established and highly standardized with regard to the use of anticoagulant, separation and processing techniques, centrifugal force, and temperature and time, resulting in highly predictable amount of cells and solid components. In an embodiment, the blood is allogeneic equine, canine, or feline blood.

[0042] The term "platelet rich plasma (PRP)" in this invention, is to be understood as a sample of blood with concentrations of platelets above baseline values measured in whole blood from a blood donor. Platelets play a key role in healing response via secretion of growth factors and recruiting reparative cells. Platelets are activated when exposed to damaged blood vessels, they aggregate at the site and form a clot. At this time they release more than 30 bioactive proteins many of have a fundamental role in hemostasis and / or tissue healing.

[0043] Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0044] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within that range, as well as the recited endpoints. The expression "% by weight", "weight percent", "%wt" or "wt%", here and throughout the description unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of the formulation.

[0045] Whereas the terms "one or more" or "at least one", such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.

[0046] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, definitions for the terms used in the description are included to better appreciate the teaching of the present invention. The terms or definitions used herein are provided solely to aid in the understanding of the invention.

[0047] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the following claims, any of the claimed embodiments can be used in any combination.

[0048] Detailed description

[0049] Platelet-rich plasma (PRP) has been clinically used as an easily prepared growth factor cocktail that can promote wound healing, angiogenesis, and tissue remodeling. Despite the increasing use of PRP therapeutically, its reported clinical effects are quite variable. And although many commercialized devices are now available for the clinical preparation of PRP, there is no standardized protocol for PRP preparation that efficiently maximizes platelet yield, platelet activation, while decreasing platelet aggregation. The current protocols often rely on calcium activation to increase the release of growth factors from the platelets. However, CaCH addition complicates handling, particularly in view of the tightening regulations for blood-derived products.

[0050] Furthermore, there remains a need in the art for an improved method for obtaining blood-derived products having an enrichment of certain beneficial proteins, such as blood-derived products having an enrichment of alpha 2 macroglobulin (02- Macroglobulin, A2MG) or Interleukin Receptor Antagonist Protein (IRAP).

[0051] The invention thereto aims to provide an optimized method for preparing blood- derived products such as PRP and samples enriched for certain beneficial proteins.

[0052] In a first aspect, the invention relates to a method for obtaining a blood-derived product, wherein said blood-derived product is chosen from platelet rich plasma (PRP), an 02-macroglobulin enriched sample or an Interleukin-1 receptor antagonist (IRAP) enriched sample, said method comprising: a. withdrawing blood from a blood donor and collecting said blood in a recipient optionally comprising an anticoagulant, thereby obtaining a whole blood sample; b. optionally performing a fractionation step on said whole blood sample, thereby obtaining a fractionated blood sample; c. optionally performing an incubation step on said whole blood sample or said fractionated blood sample, wherein said incubation step is performed for at least 30 minutes at a temperature between 28-41°C; d. optionally performing a fractionation step on said incubated sample.

[0053] The blood-derived product obtained by the method of the current invention is prepared from whole blood. In a preferred embodiment, said whole blood comprises venous blood.

[0054] Said blood donor can be any suited mammalian donor. The blood may originate from all mammals, including, but not limited to, humans, domestic and farm animals, zoo animals, sport animals, pet animals, companion animals and experimental animals, such as, for example, mice, rats, rabbits, dogs, cats, cows, horses, pigs and primates, e.g., monkeys and apes; especially horse, human, cat, dogs, rodents, etc. In an embodiment, said blood donor is an equine, which allows multiple blood collections per year with minimal discomfort or morbidities for the donor animal. Said equine may be any horse-like animal of the Equidae family, preferably of the genus Equus, such as from the species E. caballus (including the myriad domestic strains), E. zebra, E. burchelli, and E. grevyi (zebras) or E. asinus and E. hemionus (wild asses). Said equine is preferably a horse of E. caballus.

[0055] In an embodiment, said whole blood is collected in a recipient comprising an anticoagulant. In an embodiment, the (inside of) the recipient is coated with an anticoagulant. Said recipient can be any recipient suited for collecting blood. In an embodiment, said recipient is a disposable biomedical transparent flexible poly vinyl chloride(PVC) container. In an embodiment, said recipient is a syringe.

[0056] Said anticoagulant can be any anticoagulant known from the art. In an embodiment, said anticoagulant comprises citrate, for instance sodium citrate. In an embodiment, said recipient is a bag comprising citrate. In an embodiment, said recipient comprises Citrate Phosphate Dextrose Adenine (CPDA). Citrate acts as anticoagulant by chelating calcium, dextrose is needed for metabolism of stored red blood cells, phosphate is needed for a lower acidity and for having a higher concentration of 2,3- diphosphoglyceric acid (2,3 DPG) and red cell phosphate and adenine improves the viability of red blood cells. In an embodiment said recipient comprises pyrophosphate-phosphate-adenine-dextrose (PPDA-1). In an embodiment said recipient comprises Acid Citrate Dextrose Solution (ACD-A).

[0057] In an embodiment, said anticoagulant is a sterile liquid solution which is added to the recipient.

[0058] Said whole blood sample can be of any size, for instance between 1-50 ml, 50-100 ml, 100-150 ml, 150-200 ml, 200-250 ml, 250-300 ml, 300-350 ml, 350-400 ml, 400-450 ml, 450-500 ml, 500-550 ml, 550-600 ml, 600-650 ml, 650-700 ml, 700- 750 ml, 750-800 ml, 800-850 ml, 850-900 ml, 900-950 ml, 950-1000 ml, 1000-1050 ml, 1050-1100 ml, 1100-1150 ml, 1150-1200 ml, 1200-1250 ml, 1250-1300 ml, 1300-1350 ml, 1350-1400 ml, 1400-1450 ml, 1450-1500 ml, 1500-1550 ml, 1550- 1600 ml, 1600-1650 ml, 1650-1700 ml, 1700-1750 ml, 1750-1800 ml, 1800-1850 ml, 1850-1900 ml, 1900-1950 ml or between 1950-2000 ml.

[0059] In an embodiment, said whole blood sample is divided in smaller samples for processing (for instance centrifugation or incubation steps), said smaller samples being collected in smaller recipients, for instance in sterile tubes for centrifugation (for instance sterile 50 ml centrifugation tubes). In an embodiment, said smaller recipient also comprises an anticoagulant. In an embodiment, said blood is divided into smaller samples using a needle, such as a 18-gauge needle. In an embodiment, said smaller recipient is a glass serum tube.

[0060] In an embodiment, said incubation step occurs in a recipient not comprising beads (for instance glass beads).

[0061] As described above, the incubation step is performed at a temperature between 28- 41°C; such as 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41°C or any value in between. In an embodiment, said incubation step is performed at a temperature between 34-40°C, such as 37°C.

[0062] In an embodiment, said incubation step is performed for a period between 30 minutes and 4 hours, such as for a period of 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 135 minutes, 150 minutes, 165 minutes, 180 minutes, 195 minutes, 210 minutes, 225 minutes, 240 minutes or any value in between. In an embodiment, said incubation step is performed for a period between 30 minutes and two hours. In an embodiment, said incubation step is performed for a period between 30 minutes and 90 minutes. In an embodiment, said incubation step is performed for a period between 45 minutes and 75 minutes, such as 45, 46,

[0063] 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,

[0064] 68, 69, 70, 71, 72, 73, 74 or 75 minutes or any value in between.

[0065] In an embodiment, said incubation step is performed for more than 4 hours, such as 24 hours. In an embodiment, said incubation step is performed for more than 5 hours. In an embodiment, said incubation step is performed for more than 6 hours. In an embodiment, said incubation step is performed for more than 7 hours. In an embodiment, said incubation step is performed for more than 8 hours. In an embodiment, said incubation step is performed for more than 9 hours. In an embodiment, said incubation step is performed for a period of 5, 6, 7, 8, 9, 10, 11,

[0066] 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32,

[0067] 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,

[0068] 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 hours or any value in between. In a further aspect, the invention relates to a blood-derived product obtainable by the method of the current invention, wherein said blood-derived product is chosen from an Interleukin-1 receptor antagonist (IRAP) enriched sample, a platelet rich plasma (PRP) and an 02-macroglobulin enriched sample. Said blood-derived products can be used in the treatment of a musculoskeletal disease, preferably a bone disease, a tendon disease or a joint disease in a subject.

[0069] Method for obtaining an IRAP enriched sample

[0070] Degenerative joint disease, also known as arthritis, is an inflammatory condition characterized by an imbalance in which cartilage is broken down faster than it can be repaired or maintained by the body. The inflammatory process causes pain through distension of the sensitive joint capsule, and also accelerates the break down process of cartilage through small proteins called inflammatory mediators. One of the most important inflammatory mediators identified is Interleukin-1 (IL-1).

[0071] Administration of Interleukin Receptor Antagonist Protein (IRAP) is a relatively new therapy that has been developed to treat musculoskeletal diseases, such as joint inflammation. This key protein prevents IL-1 from binding to cells within the joint and exerting its degradative effects.

[0072] The current invention provides an improved method for obtaining blood-derived products having an enrichment of Interleukin Receptor Antagonist Protein (IRAP).

[0073] The inventors discovered that the method of the current invention, more specifically, performing a specific incubation step on a whole blood sample, allows to obtain an Interleukin-1 receptor antagonist (IRAP) enriched sample. An "Interleukin-1 receptor antagonist (IRAP) enriched sample" as used herein refers to a sample having a higher concentration of IRAP compared to the concentration of IRAP in whole blood without any further processing step.

[0074] In a preferred embodiment, in a method for obtaining an IRAP enriched sample said whole blood sample is collected and incubated in a recipient which does not comprise an anticoagulant, nor a clot activator. In a preferred embodiment, said incubation step occurs in a recipient not comprising beads (for instance glass beads). In an embodiment, the whole blood sample is incubated for more than 4 hours, such as 24 hours. In an embodiment, the whole blood sample is incubated for more than 5 hours. In an embodiment, the whole blood sample is incubated for more than 6 hours. In an embodiment, the whole blood sample is incubated for more than 7 hours. In an embodiment, the whole blood sample is for more than 8 hours. In a preferred embodiment, the whole blood sample is incubated for more than 9 hours.

[0075] In a preferred embodiment, the whole blood sample is incubated between 22 and 26 hours, such as 24 hours, at a temperature between 34-40°C, such as 37°C. This incubation step allows the white blood cells to produce IRAP and release the intracellular protein into the blood sample. Blood storage temperature as well as time before first centrifugation are important contributors to plasma proteome variability. Intracellular protein release occurs when plasma remains in contact with blood cells (which is the case during incubation of whole blood samples) for prolonged periods. In a preferred embodiment, said incubation step occurs in a recipient not comprising beads (for instance glass beads).

[0076] In a preferred embodiment, the whole blood sample is incubated at a temperature between 28-41°C within 2 hours after withdrawing blood from a blood donor. As such, the decrease in temperature of the blood sample after withdrawing from the donor and before incubation is limited. In an embodiment, the decrease in temperature is at most 5°C, preferably at most 2°C, before the incubation step occurs.

[0077] In a preferred embodiment, the method further comprises a fractionation step on said incubated sample. More specifically, after said incubation step, the serum containing IRAP is preferably separated from the blood cells by centrifugation. In a further embodiment, after said centrifugation step, the serum containing IRAP is further subjected to a filtration step.

[0078] Said IRAP protein concentration in said sample can be determined by any suitable technique known from the art for measuring a protein concentration, such as an immuno-enzymatic method, an immuno-histochemical method or mass spectrometry. In an embodiment, said IRAP protein concentration in said sample is determined by means of ELISA (Enzyme Linked Immunosorbent Assay) or quantitative mass spectrometry (for instance LC-MS / MS shotgun proteomic analysis). In an embodiment, said IRAP messenger RNA (mRNA) concentration in said sample can be determined by any suitable technique known from the art for measuring a mRNA concentration, such as for instance reverse transcription polymerase chain reaction (RT-PCR).

[0079] In a preferred embodiment, said IRAP enriched sample comprises at least 10 ng / ml Interleukin-1 receptor antagonist (IRAP).

[0080] In a further aspect, the invention relates to a blood-derived product obtainable by above described method wherein an incubation step is performed on a whole blood sample, optionally followed by a fractionation step on said incubated sample. Said blood-derived product obtained by said method is an Interleukin-1 receptor antagonist (IRAP) enriched sample. In a preferred embodiment, said IRAP enriched sample comprises at least 10 ng / ml Interleukin-1 receptor antagonist (IRAP), such as 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, 20 ng / ml, 21 ng / ml, 22 ng / ml, 23 ng / ml, 24 ng / ml, 25 ng / ml,

[0081] 26 ng / ml, 27 ng / ml, 28 ng / ml, 29 ng / ml, 30 ng / ml, 31 ng / ml, 32 ng / ml, 33 ng / ml,

[0082] 34 ng / ml, 35 ng / ml, 36 ng / ml, 37 ng / ml, 38 ng / ml, 39 ng / ml, 40 ng / ml, 41 ng / ml,

[0083] 42 ng / ml, 43 ng / ml, 44 ng / ml, 45 ng / ml, 46 ng / ml, 47 ng / ml, 48 ng / ml, 49 ng / ml,

[0084] 50 ng / ml or any value in between. In an embodiment, said IRAP enriched sample comprises at least 50 ng / ml IRAP, such as 50 ng / ml, 55 ng / ml, 60 ng / ml, 65 ng / ml,

[0085] 70 ng / ml, 75 ng / ml, 80 ng / ml, 85 ng / ml, 90 ng / ml, 95 ng / ml, 100 ng / ml or any value in between. In an embodiment, said IRAP enriched sample comprises at least 100 ng / ml, such as 100 ng / ml, 125 ng / ml, 150 ng / ml, 175 ng / ml, 200 ng / ml, 225 ng / ml, 250 ng / ml, 275 ng / ml, 300 ng / ml, 325 ng / ml, 350 ng / ml, 375 ng / ml, 400 ng / ml, 425 ng / ml, 450 ng / ml, 475 ng / ml, 500 ng / ml, 525 ng / ml, 550 ng / ml, 575 ng / ml, 600 ng / ml, 625 ng / ml, 650 ng / ml, 675 ng / ml, 700 ng / ml, 725 ng / ml, 750 ng / ml, 775 ng / ml, 800 ng / ml, 825 ng / ml, 850 ng / ml, 875 ng / ml, 900 ng / ml, 925 ng / ml, 950 ng / ml, 975 ng / ml, 1000 ng / ml, 1025 ng / ml, 1050 ng / ml, 1075 ng / ml,

[0086] 1100 ng / ml, 1125 ng / ml, 1150 ng / ml, 1175 ng / ml, 1200 ng / ml, 1225 ng / ml, 1250 ng / ml, 1275 ng / ml, 1300 ng / ml, 1325 ng / ml, 1350 ng / ml, 1375 ng / ml, 1400 ng / ml, 1425 ng / ml, 1450 ng / ml, 1475 ng / ml or 1500 ng / ml Interleukin-1 receptor antagonist (IRAP) or any value in between.

[0087] In an embodiment, said IRAP enriched sample comprises between 10-100 ng / ml, 100-200 ng / ml, 300-400 ng / ml, 400-500 ng / ml, 500-600 ng / ml, 600-700 ng / ml, 700-800 ng / ml, 800-900 ng / ml, 900-1000 ng / ml, 1000-1100 ng / ml, 1100-1200 ng / ml, 1200-1300 ng / ml, 1300-1400 ng / ml, 1400-1500 ng / ml, 1500-1600 ng / ml, 1600-1700 ng / ml, 1700-1800 ng / ml, 1800-1900 ng / ml, 1900-2000 ng / ml, 2000- 2100 ng / ml, 2100-2200 ng / ml, 2200-2300 ng / ml, 2300-2400 ng / ml or between 2400-2500 ng / ml Interleukin-1 receptor antagonist (IRAP).

[0088] In a preferred embodiment, IL-ip levels in said IRAP enriched sample are not increased compared to a serum control sample. In a preferred embodiment, IL-ip levels in said IRAP enriched sample are at most 3 fold, more preferably at most 2 fold, more preferably at most 1.5 fold increased compared to a serum control sample.

[0089] In an embodiment, the IRAP / IL-ip ratio in said IRAP enriched sample is higher than 2, more preferably higher than 3, more preferably higher than 4, more preferably higher than 5, more preferably higher than 6, more preferably higher than 7, more preferably higher than 8, more preferably higher than 9, more preferably higher than 10, more preferably higher than 11, more preferably higher than 11, more preferably higher than 12, more preferably higher than 13, more preferably higher than 14, more preferably higher than 15.

[0090] Such an IRAP enriched sample can be used for the treatment of musculoskeletal diseases (as discussed in more detail below).

[0091] Method for obtaining a PRP sample

[0092] Platelet-rich plasma (PRP) has been clinically used as an easily prepared growth factor cocktail that can promote wound healing, angiogenesis, and tissue remodeling. It was revealed through a number of studies that various bioactive substances, including platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-p), and epidermal growth factor (EGF), were discharged from the a-granules of platelets into plasma when the platelets were destroyed and activated.

[0093] All of these growth factors have been evaluated for their ability to enhance tenocyte mitogenesis and synthesis of matrix molecules such as collagen types l and III. These PRP containing growth factors have also been extensively investigated in the light of cartilage repair in degenerative joint disease. Specifically, they promote stromal stem cell proliferation and angiogenesis and are regarded as key signals in tissue repair / regeneration.

[0094] Despite the increasing use of PRP therapeutically, its reported clinical effects are quite variable. Although many commercialized devices are now available for the clinical preparation of PRP, there is no standardized protocol for PRP preparation. There remains a need in the art to provide a method for optimizing the preparation of PRP. For example, it has been shown that the centrifugal force exerted on whole blood contained in common laboratory ware affects PRP yield. Similarly, the complexity of blood coagulation (fibrin polymerization) and platelet aggregation further complicates the preparation of PRP.

[0095] The method of the current invention optimizes the protocol for preparing PRP. This entails the establishment of a reliable and practical method to maximize platelet yield and concentration in plasma using common laboratory ware and equipment.

[0096] In a preferred embodiment, said whole blood sample is collected in a recipient comprising an anticoagulant. In an embodiment, the (inside of) the recipient is coated with an anticoagulant. Said recipient can be any recipient suited for collecting blood. Suitable coagulants are described above and include for instance ACD-A and citrate.

[0097] In a preferred embodiment, no platelet activator (such as CaCk, thrombin, collagen,...) is added.

[0098] In a preferred embodiment, said platelets are activated by one or more freeze-drying steps. In an embodiment, said one or more freeze-drying steps are performed at - 20°C. In an embodiment, said one or more freeze-drying steps are performed at -20 °C for a duration of 8 to 24 hours, preferably between 12 and 18 hours, depending on the volume and moisture content of the platelet preparation. The duration may be adjusted to ensure complete sublimation of water without denaturation of the platelet-derived bioactive factors.

[0099] The inventors discovered that the method of the current invention, more specifically, performing a specific incubation step on a fractioned blood sample (wherein the blood sample undergoes one or more centrifugation steps at a centrifugal force between 100 and 1000 g or is allowed to sediment for 2 hours at room temperature (RT= between 20°C and 25°C)) allows to obtain a PRP sample.

[0100] Blood fractionation is the process of fractionating whole blood, or separating it into its component parts. This is typically done by centrifuging the blood.

[0101] In an embodiment, the resulting components are: a clear solution of blood plasma in the upper phase (which can be separated into its own fractions), the buffy coat, which is a thin layer of leukocytes (white blood cells) mixed with platelets in the middle, and erythrocytes (red blood cells) at the bottom of the centrifuge tube.

[0102] In an embodiment, said fractionated blood sample is obtained by one or more centrifugation steps at a centrifugal force between 100 and 1000 g, for instance at a centrifugal force between 100-200 g, 200-300 g, 300-400 g, 400-500 g, 500-600 g, 600-700 g, 700-800g, 800-900 g, 900-1000 g. In an embodiment, said fractionated blood sample is obtained by one or more centrifugation steps at a centrifugal force of 100 g, 150 g, 200 g, 250 g, 300 g, 350 g, 400 g, 450 g, 500 g, 550 g, 600 g, 650 g, 700 g, 750 g, 800 g, 850 g, 900 g, 950 g, 1000 g or any value in between.

[0103] In an embodiment, said fractionated blood sample is obtained by one centrifugation step at a centrifugal force between 100 and 1000 g, for instance at a centrifugal force between 100-200 g, 200-300 g, 300-400 g, 400-500 g, 500-600 g, 600-700 g, 700- 800g, 800-900 g or between 900-1000 g. In an embodiment, said fractionated blood sample is obtained by one centrifugation step at a centrifugal force of 100 g, 150 g, 200 g, 250 g, 300 g, 350 g, 400 g, 450 g, 500 g, 550 g, 600 g, 650 g, 700 g, 750 g, 800 g, 850 g, 900 g, 950 g or 1000 g or any value in between.

[0104] In an embodiment, said fractionated blood sample is obtained by more than one centrifugation step at a centrifugal force between 100 and 1000 g. In an embodiment, said fractionated blood sample is obtained by two, three, four, five, six, seven, eight, nine or ten centrifugation steps at a centrifugal force between 100 and 1000 g. In a preferred embodiment, said fractionated blood sample is obtained by two centrifugation steps at a centrifugal force between 100 and 1000 g.

[0105] In an embodiment, said multiple centrifugation steps are performed at the same centrifugal force, for instance at a centrifugal force between 100-200 g, 200-300 g, 300-400 g, 400-500 g, 500-600 g, 600-700 g, 700-800g, 800-900 g, 900-1000 g. In an embodiment, said fractionated blood sample is obtained by more than one centrifugation step at the same centrifugal force of 100 g, 150 g, 200 g, 250 g, 300 g, 350 g, 400 g, 450 g, 500 g, 550 g, 600 g, 650 g, 700 g, 750 g, 800 g, 850 g, 900 g, 950 g, 1000 g or any value in between. In a preferred embodiment, said fractionated blood sample is obtained by two centrifugation steps at a centrifugal force of 200 g.

[0106] In an alternative embodiment, said multiple centrifugation steps are performed at a different centrifugal force between 100 and 1000 g. In an embodiment, two centrifugation steps are performed at a different centrifugal force between 100 and 1000 g. In a further embodiment, said first centrifugation step is performed at a lower centrifugal force than said second centrifugation step. In an alternative further embodiment, said first centrifugation step is performed at a higher centrifugal force than said second centrifugation step.

[0107] In an alternative embodiment, a specific incubation step on a fractioned blood sample is performed, wherein said fractionated blood sample is obtained by allowing the blood sample to sediment for 2 hours at room temperature (RT, meaning between 20-25°C).

[0108] After a first centrifugation step, the resulting supernatant comprising platelets is collected. In an embodiment, said resulting supernatant is further processed by means of one or more centrifugation steps. In a preferred embodiment, said supernatant is subjected to a second centrifugation step to form a pellet of residual red blood cells at the bottom of the centrifugation tube.

[0109] According to the current invention, after said fractionation step (in this case one or more centrifugation steps at a centrifugal force between 100 and 1000 g or a sedimentation step for 2 hours at room temperature), the fractionated sample is incubated for at least 30 minutes at a temperature between 28-41°C, which allows to obtain a platelet rich plasma sample comprising at least 50X109platelets / l iter after said incubation step.

[0110] There are many different methods for the preparation of a PRP, which differ in speed, time, and number of centrifugation steps, blood volume used, anticoagulative supplementation, and activation / incubation methods.

[0111] In a preferred embodiment, at least two centrifugation steps are performed. In an embodiment, one or more centrifugation steps at 200 g are followed by a centrifugation step at 1000 g. As described above, various bioactive substances, including platelet-derived growth factor (PDGF), transforming growth factor-beta 1 (TGF- 1), and epidermal growth factor (EGF), were discharged from the a-granules of platelets into plasma when the platelets were destroyed and activated. Activation of such growth factor release from platelet concentrates prior to clinical application has frequently been described, especially for PRP production. The method of the current invention is advantageous, as it allows activation of the platelets without the addition of calcium. This is beneficial, given that CaC addition complicates handling, particularly in view of the tightening regulations for blood-derived products. Lowering of the centrifugation speed and performing an incubation step incubated for at least 30 minutes at a temperature between 28-41°C according to the method of the current invention, results in a better activation of the platelets and a higher release of the desired growth factors.

[0112] In an embodiment, the method further comprises one or more freeze-thaw cycles to lyse the platelets, thereby releasing various bioactive substances, such as the growth factors described above. In embodiments, the method comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 freeze-thaw cycles.

[0113] In an embodiment, the PRP sample can be lysed to obtain a platelet lysate. In an embodiment, one or more freeze-thaw cycles are performed to lyse the platelets in the PRP sample. In an embodiment, at least one freeze-thaw cycle is performed in - 20°C. In an embodiment, more than one freeze-thaw cycle (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 freeze-thaw cycles) is performed in -20°C.

[0114] In a preferred embodiment, the method encompasses the following steps. Venous blood (preferably from an equine donor) is collected in a citrate bag and further divided in sterile 50 ml centrifugation tubes, which are centrifuged at 200 g for 15 minutes at room temperature with the centrifuge brake off. After this treatment two phases are obtained: a dark one constituted by precipitated red and white cells at the bottom of the tubes, and a clear one visible in the upper part of the probe, consisting of platelet rich plasma. This plasma is taken (for instance by means of serological pipet) and transferred into other centrifugation tubes, which are again centrifuged at 200 g for 5 minutes at room temperature with the centrifuge brake off. Thanks to this centrifugation step a pellet of residual red blood cells is formed at the bottom of the tube. In a next step the supernatant is collected in other sterile 50ml tubes. Said supernatant comprises the platelet rich plasma (PRP) product, which is incubated for at least 30 minutes at a temperature between 28-41°C. After this incubation step, the resulting product can for instance be further aliquoted in doses suitable for injection. In an embodiment, 1 ml of said incubated supernatant (the PRP product) can be aliquoted in a 2 ml cryotube. This method allows to recover at least 80% of platelets present in whole blood.

[0115] In a further aspect, the invention relates to a blood-derived product obtainable by above described method wherein an incubation step is performed on a fractionated sample, said fractionated sample being obtained by one or more centrifugation steps at a centrifugal force between 100 and 1000 g. Said blood-derived product obtained by said method is a PRP sample.

[0116] In a preferred embodiment, said thus obtained PRP sample comprises at least 30xl09platelets / liter, more preferably at least 40X109platelets / liter, more preferably at least 50X109platelets / liter. In an embodiment, said thus obtained PRP sample comprises at least 60X109platelets / liter, at least 70X109platelets / liter, at least 80X109platelets / liter, at least 90X109platelets / liter, at least 100X109platelets / liter (lOOxlO6platelets / ml), such as 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200X109platelets / liter. In an embodiment, said thus obtained PRP sample comprises at most 200xl09platelets / liter. In a preferred embodiment, said thus obtained PRP sample comprises between 70% and 100% platelets, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% platelets or any value in between, wherein the sum of the white blood cells, red blood cells and platelets in the sample represents 100%. In a preferred embodiment, said thus obtained PRP sample comprises between 90-100% nonaggregated platelets. In an embodiment, the main platelet volume of said platelets in said thus obtained PRP sample is below 20 femtoliters (fL), more preferably below 10 fL (for instance measured by a Coulter Counter). In an embodiment, the platelet distribution width (platelet function / activation marker) of said platelets in said thus obtained PRP sample is between 10 and 20%. In an embodiment, the procalcitonin percentage in said thus obtained PRP sample is between 0.005 and 0.1%.

[0117] In a preferred embodiment, said thus obtained PRP sample comprises at least 50 ng / ml insulin-like growth factor (IGF)-I. In embodiments, said thus obtained PRP sample comprises at least 55 ng / ml, 60 ng / ml, 65 ng / ml, 70 ng / ml, 75 ng / ml, 80 ng / ml, 85 ng / ml, 90 ng / ml, 95 ng / ml, 100 ng / ml, 105 ng / ml, 110 ng / ml, 115 ng / ml, 120 ng / ml, 125 ng / ml, 130 ng / ml, 135 ng / ml, 140 ng / ml, 145 ng / ml, 150 ng / ml, 155 ng / ml, 160 ng / ml, 165 ng / ml, 170 ng / ml, 175 ng / ml, 180 ng / ml, 185 ng / ml, 190 ng / ml, 195 ng / ml or 200 ng / ml.

[0118] In an embodiment, said thus obtained PRP sample comprises at least a 1.5 fold increase in insulin-like growth factor (IGF)-I compared to baseline. In embodiments, said thus obtained PRP sample comprises at least a 2.0, 2.5 or 3.0 fold increase in IGF-I compared to baseline. As used herein, baseline can refer to a blood sample or a plasma sample.

[0119] In the context of joint biology, IGF-I is considered to be an essential anabolic growth factor in the regulation of cartilage metabolism and exerts its effects by binding to the IGF-I type 1 receptor on the chondrocyte membrane (discussed in more detail below).

[0120] In a preferred embodiment, said thus obtained PRP sample comprises a concentration of IGF-I between 50-1000 ng / ml, more preferably between 50 and 500 ng / ml, such as between 80-140 ng / ml.

[0121] White blood cells may pose a concern in tissue regenerative efforts due to the pro- inflammatory mediators such as neutral proteases and acid hydrolases contained in white blood cells. This finding is of clinical importance when considering the pro- inflammatory cytokines contained in mononuclear white blood cells, and their potential for inciting an undesirable inflammatory reaction in an environment such as a tendon or joint.

[0122] As such, in a preferred embodiment, said PRP sample comprises less than 1%, preferably less than 0.5% white blood cells, wherein the sum of the white blood cells, red blood cells and platelets in the sample represents 100%. In a preferred embodiment, said PRP sample comprises less than 500 white blood cells / pl.

[0123] Other impurities include red blood cells. In a preferred embodiment, said PRP sample comprises less than 29% red blood cells. In a preferred embodiment, said PRP sample comprises even less red blood cells, such as less than 25%, less than 20%, less than 15%, less than 10 %, less than 5% or less than 1% red blood cells. In a preferred embodiment, said PRP sample comprises less than 30xl03red blood cells / pl. In a preferred embodiment, the variation in size of said red blood cells in said PRP sample is minimal. In a preferred embodiment, said red cell distribution width of said red blood cells in said PRP sample is less than 1%, more preferably less than 0.5%.

[0124] In a preferred embodiment, said PRP sample comprises less than 1% granulocytes, more preferably less than 0.5% granulocytes.

[0125] Method for obtaining an a2-macrocjlobulin enriched sample

[0126] Alpha 2 macroglobulin (A2MG) is a potent, naturally occurring protease inhibitor that is constitutively present in blood and at lesser levels in normal synovial fluid and cartilage of various vertebrate species, including horses. A2MG has inhibitory activity against all degradative endoprotease classes, particularly those activated by acute inflammation. This broad spectrum effect is a result of the A2MG molecular structure, which has been described as a unique bait-and-trap mechanism.

[0127] In addition to protease neutralization, A2MG binds to proinflammatory cytokines to reduce cytokine-induced synthesis of collagenases in cartilage. Thus, A2MG has two principal cartilage-sparing effects: binding to proinflammatory cytokines that initiate the process of cartilage degradation and neutralizing catabolic enzymes that drive the progression of osteoarthritis (OA).

[0128] Insulin-like growth factors (IGFs) are crucial for many aspects of development, growth, and metabolism. IGF-I, a 70-amino-acid single-chain polypeptide, is a key cell survival factor regulating somatic growth and cellular proliferation. Most of its mitogenic and metabolic responses are mediated via the IGF type I receptor, a receptor tyrosine kinase expressed in many cell types. Receptor engagement and subsequent activation are determined by the availability of free IGF-I. However, the majority of the circulating IGF-I pool is found in high-affinity complexes with IGF- binding proteins (IGFBPs). Thus, the serum half-life, tissue distribution, and biological availability of free IGF-I are predominantly modulated by the IGFBPs. In the context of joint biology, IGF-I is considered to be an essential anabolic growth factor in the regulation of cartilage metabolism and exerts its effects by binding to the IGF-I type 1 receptor on the chondrocyte membrane. Mechanisms for controlling the effects of IGF-I include alterations in the level of this growth factor, its receptor and / or the IGF-I affinity or availability to its receptor. Disturbance of any one of the above elements may induce a disregulation of the mechanisms involved in the local control of joint tissue integrity. Recent research has focused on application of IGF-I and / or interleukin-I (IL-I) receptor antagonist gene therapy for cartilage repair in horse. In the context of bone metabolism, IGF-I, in cooperation with others growth factors such as transforming growth factor p and fibroblast growth factor, plays a significant role in the remodelling process of bone and has a favourable impact on healing of fractures. Systemic factors that are taken up by the osseous tissue as well as local factors with a paracrine / autocrine function produced by the osteoblast are involved. At the level of the growth plate, many growth factors such as IGF-I, but also Indian hedgehog, PTHrP, fibroblast growth factors, bone morphogenetic proteins and vascular endothelial growth factor, are now considered crucial regulators of chondrocyte proliferation and differentiation. These growth factors are implicated in the development orthopedic diseases.

[0129] A2MG is a binding protein of IGFBP-1 resulting in enhanced IGF effects.

[0130] The current invention provides a method for obtaining an A2MG-enriched sample.

[0131] Said whole blood sample is collected in a recipient comprising an anticoagulant. Said recipient can be any recipient suited for collecting blood. Suitable coagulants are described above and include for instance ACD-A and citrate. Said recipient can for instance be a syringe, such as a 50 ml syringe.

[0132] In a next step, said collected whole blood sample is subjected to one or more separation steps to obtain a plasma sample.

[0133] Separation steps which allow to obtain a plasma sample and can be used in the method of the current invention for instance include centrifugation, gravitational sedimentation, membrane filtration, sedimentation using a sedimentation cone, sedimentation in a capillary or elongated sedimentation tube, plasma skimming via a density barrier, microfluidic separation via hydrodynamic flow, porous membrane- assisted separation, use of a plasma separation membrane impregnated with anticoagulant, passive sedimentation over a defined period of time.

[0134] In an embodiment, said one or more separation steps comprise subjecting said collected whole blood sample to one or more centrifugation steps to obtain a plasma sample. In an embodiment, said one or more separation steps does not comprise subjecting said collected whole blood sample to one or more centrifugation steps to obtain a plasma sample.

[0135] Plasma may be obtained from whole blood without the use of centrifugation by employing various alternative separation methods. One such method is gravitational sedimentation, in which the whole blood is allowed to rest under the influence of gravity so that the denser cellular components settle at the bottom of the container while a plasma-rich layer forms at the top. In another approach, membrane filtration is used, wherein the blood is passed through a semi-permeable membrane that permits plasma to pass through while retaining cellular components such as red and white blood cells and platelets. Plasma may also be obtained by introducing the blood into a sedimentation cone, where the conical geometry enhances the settling of blood cells and facilitates the collection of plasma from the upper portion. Similarly, sedimentation can be performed in a capillary or elongated sedimentation tube, where the high height-to-diameter ratio promotes faster and more distinct separation of plasma from cells. Plasma skimming may further be performed by removing the plasma layer after partial sedimentation, optionally across a density barrier that minimizes mixing between layers. More advanced techniques may include microfluidic separation via hydrodynamic flow, in which fluidic forces and the design of microchannels guide cellular elements away from the plasma collection path, enabling continuous separation. Porous membrane-assisted separation may also be employed, whereby whole blood is placed against a membrane with pore sizes selected to allow the passage of plasma while excluding blood cells. In some embodiments, the membrane may be impregnated with an anticoagulant such as heparin or EDTA, which prevents coagulation and supports plasma migration through the membrane. Finally, plasma may be obtained by passive sedimentation over a defined period of time, for instance several hours, during which plasma naturally separates from the cellular fraction in a static container without mechanical intervention.

[0136] In an embodiment, said one or more separation steps comprise subjecting said collected whole blood sample to gravitational sedimentation and / or membrane filtration to obtain a plasma sample.

[0137] When plasma is obtained from whole blood by sedimentation under gravity, the duration required for sufficient separation depends on various factors, including the hematocrit level, temperature, anticoagulant type, and geometry of the sedimentation vessel. Typically, a visible separation between the plasma and cellular components can be observed after approximately 1 to 2 hours at room temperature. In certain embodiments, sedimentation may be carried out over a period ranging from 4 to 12 hours. For applications requiring high purity of the plasma fraction, or when using blood with a high hematocrit value, the sedimentation process may be extended to 24 hours or more. In preferred embodiments, sedimentation is conducted at a controlled temperature between 4 °C and 25 °C to ensure sample stability and minimize hemolysis. The sedimentation time may also be adjusted based on the shape and size of the vessel, with conical or elongated tubes enabling more efficient separation within shorter time frames.

[0138] In an embodiment, said whole blood sample is transferred to one or more separator tubes prior to said one or more separation steps.

[0139] In a preferred embodiment, said one or more separator tubes comprise a clot activator. Common clot activators include silica particles (silicon dioxide), which are typically coated on the interior walls of the tube to initiate the intrinsic coagulation pathway. In some configurations, the clot activator may consist of finely divided glass particles or microparticles that likewise promote clot formation upon contact with whole blood. Alternatively, thrombin may be employed as a clot activator to accelerate the coagulation process. The clot activator may be used alone or in combination with a gel separator, as is the case in serum separator tubes (SST), which combine silica-based activators with polymeric gel barriers to facilitate separation of serum following centrifugation.

[0140] In an embodiment, said one or more separator tubes comprise a septum cap, which is a sealing element designed to permit the sterile introduction and withdrawal of fluids without necessitating the removal of the cap itself. The septum cap may vary in configuration, material, and number of septa, depending on the intended application.

[0141] The septum cap may be formed from elastomeric materials such as medical-grade silicone, butyl rubber, or thermoplastic elastomers, optionally coated with a thin layer of fluoropolymer to reduce protein adhesion and improve chemical resistance. The cap may comprise a single septum, dual septa, or even multiple discrete septa integrated into a single cap body. In a preferred embodiment, the septum cap comprises more than one septum, thereby enabling distinct pathways for sample introduction and sample extraction. For example, a first sample— such as whole blood— may be injected through the first septum using a sterile syringe or pipette. After centrifugation, the processed sample— such as plasma, serum, or a supernatant— can then be withdrawn through a second septum without breaching the sterile barrier of the tube. This configuration facilitates closed-loop sample processing and reduces the risk of cross-contamination or loss of sample integrity.

[0142] These septa may be arranged concentrically, linearly, or in a polygonal configuration, depending on user needs. The septa may have different durometers to accommodate various needle gauges or automated pipette tips, with resealing capability after puncture to maintain a sterile barrier.

[0143] The use of a septum cap significantly enhances sterility by eliminating the need to remove the cap, which could expose the interior of the separator tube to airborne contaminants or accidental contact. The septa act as self-sealing membranes, allowing repeated puncture with minimal risk of microbial ingress. This design supports aseptic workflows and is particularly advantageous in clinical, laboratory, or biotechnological settings where sterility is paramount.

[0144] In an embodiment, the method of the current invention provides an A2MG-enriched sample by means of incubation of a fractionated blood sample, more specifically by means of incubation of a filtrated blood sample.

[0145] In an embodiment, said fractionated blood sample is incubated for at least 30 minutes at a temperature between 28-41°C, for instance for 1 hour at 37°C.

[0146] In another embodiment, the method of the current invention provides an A2MG- enriched sample without an incubation step on said fractionated blood sample.

[0147] An "a2-macroglobulin (A2MG) enriched sample" as used herein refers to a sample having a higher concentration of 02-macroglobulin compared to the concentration of 02-macroglobulin in whole blood without any further processing step. More specifically, in the current invention, said fractionated blood sample is obtained by filtration using a molecular weight cut-off membrane, thereby obtaining a concentrated plasma sample.

[0148] Molecular Weight Cut-Off (MWCO) is a specification of membrane filters that defines the smallest molecular weight of a solute (typically in Daltons) that the membrane can retain effectively. For example: a 100 kDa MWCO membrane is designed to retain molecules with a molecular weight > 100,000 Daltons. Smaller molecules, like salts or small proteins, will pass through, while larger molecules (e.g. A2MG, which is around 720 kDa as a tetramer) will be retained.

[0149] In a preferred embodiment, said collected whole blood sample is first subjected to one or more centrifugation steps to obtain a plasma sample (the resulting supernatant of the centrifugation step), which can be subjected to said filtration step using a molecular weight cut-off membrane of for instance more than 100 kDa to obtain a concentrated plasma sample.

[0150] Said filtration step comprises the use of a concentrator column to be used in combination with centrifugation. The membrane of this concentrator column allows smaller molecules to pass through into the collection tube, while retaining larger protein molecules above the membrane. In an embodiment, said concentrator column is disposable. In an embodiment, said concentrator column comprises a Polyethersulfone (PES) membrane. A PES membrane has low protein binding properties, high flow rates, and shows chemical resistance. Such a concentrator column works in combination with centrifugation to effectively concentrate proteins from dilute solutions.

[0151] During centrifugation the A2MG enriched plasma is retained above the membrane.

[0152] The inventors found that higher concentrations of A2MG can be obtained in the concentrated plasma sample by using a concentrator column having a filter membrane with a conical shape (see for instance number 2 on Figure 4).

[0153] The use of a conical filter membrane in a concentrator column offers several technical advantages that improve the efficiency and reliability of the concentration process. Firstly, the conical geometry inherently increases the effective filtration surface area within a given volume compared to a flat or cylindrical membrane. This increased surface area facilitates a higher throughput of fluid, reduces the likelihood of membrane fouling, and contributes to a more uniform distribution of pressure across the membrane surface, thereby enhancing overall filtration performance.

[0154] Secondly, the conical shape positively influences the fluid dynamics within the concentrator column. The tapered design promotes directional flow and minimizes stagnation zones, which are areas where fluid movement may be restricted in conventional geometries. This improved flow behavior not only results in more consistent filtration but also creates shear conditions that help prevent the aggregation or denaturation of sensitive biomolecules, such as Alpha-2- macroglobulin (A2MG), during the concentration process.

[0155] Furthermore, the conical configuration supports higher concentration factors by enabling more efficient removal of permeate, leading to shorter processing times. The geometry also assists in minimizing sample loss, as the design directs the retained concentrate toward a narrower collection zone, thereby facilitating easy and complete recovery of the target molecule.

[0156] In an embodiment, said fractionated blood sample is a concentrated plasma sample obtained by filtration using a molecular weight cut-off membrane of more than 50 kDa, thereby retaining proteins having a size larger than 50 kDa. In a preferred embodiment, said fractionated blood sample is a concentrated plasma sample obtained by filtration using a molecular weight cut-off membrane of more than 100 kDa, thereby retaining proteins having a size larger than 100 kDa. In an embodiment, said fractionated blood sample is a concentrated plasma sample obtained by filtration using a molecular weight cut-off membrane of 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa or 200 kDa, thereby retaining proteins having a size larger than 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa or 200 kDa, respectively. In an embodiment, said fractionated blood sample is a concentrated plasma sample obtained by filtration using a molecular weight cut-off membrane of more than 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa or 700 kDa, thereby retaining proteins having a size larger than 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa or 700 kDa, respectively. In an embodiment, said fractionated blood sample is a concentrated plasma sample obtained by filtration using a molecular weight cut-off membrane of more than 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa, 1050 kDa, 1100 kDa, 1150 kDa or 1200 kDa, thereby retaining proteins having a size larger than 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa, 1050 kDa, 1100 kDa, 1150 kDa or 1200 kDa, respectively.

[0157] In a preferred embodiment, said fractionated blood sample is a concentrated plasma sample obtained by filtration using a molecular weight cut-off membrane of more than 100 kDa.

[0158] As described above, in a preferred embodiment, before said filtration step using a molecular weight cut-off membrane of more than 100 kDa is performed, said collected whole blood sample is first subjected to one or more centrifugation steps to obtain a plasma sample.

[0159] In an embodiment, said collected whole blood sample is first transferred to one or more separator tubes before being subjected to said one or more separation steps. Preferably said separator tubes are not glass tubes, but plastic tubes suited for centrifugation (for instance centrifugation tubes formed from a thermoplastic polymer, such as polypropylene or polycarbonate). Such plastic tubes facilitate white blood cell adherence.

[0160] In an embodiment, said collected whole blood sample is first subjected to one or more centrifugation steps at a centrifugal force between 500 and 2500 g, more preferably between 1500 and 2500g. In an embodiment, said collected whole blood sample is first subjected to one or more centrifugation steps at a centrifugal force between 500 and 600 g, 600 and 700 g, 700 and 800 g, 800 and 900 g, 900 and 1000 g, 1000 and 1100 g, 1100 and 1200 g, 1200 and 1300 g, 1300 and 1400 g, 1400 and 1500 g, 1500 and 1600 g, 1600 and 1700 g, 1700 and 1800 g, 1800 and 1900 g, 1900 and 2000 g, 2000 and 2100 g, 2100 and 2200 g, 2200 and 2300 g, 2300 and 2400 g, or between 2400 and 2500 g.

[0161] In an embodiment, said collected whole blood sample is first subjected to one centrifugation step at a centrifugal force between 1000 and 2200 g, more preferably between 1500-2000 g, for instance at a centrifugal force between 1000-1200 g, 1200-1400 g, 1400-1600 g, 1600-1800g, 1800-2000 g, 2000-2200 g to obtain a plasma sample. In an embodiment, said collected whole blood sample is first subjected to one centrifugation step at a centrifugal force of 1000 g, 1050 g, 1100 g, 1150 g, 1200 g, 1250 g, 1300 g, 1350 g, 1400 g, 1450 g, 1500 g, 1550 g, 1600 g, 1650 g, 1700 g, 1750 g, 1800 g, 1850 g, 1900 g, 1950 g, 2000 g, 2050 g, 2100 g, 2150 g, 2200 g or any value in between. In an embodiment, said centrifugation step lasts between 5 and 60 minutes, preferably between 5 and 15 minutes, such as 10 minutes.

[0162] In a preferred embodiment, said collected whole blood sample is first subjected to one centrifugation step at a centrifugal force between 1500 and 2000 g for 10 minutes, before said filtration step using a molecular weight cut-off membrane of more than 100 kDa is performed.

[0163] In an embodiment, said collected whole blood sample is first subjected to more than one centrifugation step at a centrifugal force between 1000 and 2200 g. In an embodiment, said more than one centrifugation steps each lasts between 5 and 60 minutes, preferably between 5 and 15 minutes, such as 10 minutes.

[0164] In a preferred embodiment, said collected whole blood sample is first subjected to one centrifugation step at a centrifugal force between 1500 and 2500 g for 10 minutes, before a filtration step using a molecular weight cut-off membrane of more than 100 kDa is performed.

[0165] In an embodiment, after said last fractionation step (in this case a filtration step using a molecular weight cut-off membrane of for instance more than 100 kDa), the fractionated sample is incubated for at least 30 minutes at a temperature between 28-41°C, which allows to obtain an O2-macroglobulin enriched sample.

[0166] In a preferred embodiment, the method encompasses the following steps. Venous blood (preferably from an equine donor) is collected in a citrate bag and further divided in sterile 50 ml centrifugation tubes. In a next step, said collected whole blood sample is first subjected to one centrifugation step between 1500-2000 g (preferably 3400 rpm) for 10 minutes, before a filtration step using a molecular weight cut-off membrane of more than 100 kDa is performed, wherein said filtration step comprises the use of a concentrator column during centrifugation of the sample at a speed / centrifugal force between 1500-2000 g (preferably 3400 rpm) for 10 minutes.

[0167] In a preferred embodiment, the method encompasses the following steps. In a first step, one or more syringes (for instance 50ml syringes) are filled with anticoagulant (for instance 5ml sterile ACD-A). A donor (for instance a horse's jugular vein) is prepared for aseptic blood collection and an amount of blood is aspirated into one or more of said (50 ml) syringes comprising anticoagulant. In a preferred embodiment, the syringes are rotated several times so that the blood is well mixed with the anticoagulant.

[0168] In a next step, one or more centrifugation steps are performed to remove blood cells from the plasma. The collected whole blood samples are transferred to one or more separator tubes having a septum cap. Blood tube holders can be placed opposite to each other in the centrifuge and the separator tubes can be placed in these holders. The separator tubes can be centrifuged at 3400 RPM (2160 g) for 10 minutes. After centrifugation, the septum cap of the one or more separator tubes is disinfected for instance with an alcohol wipe. The obtained plasma can be aspirated from these separator tubes into a (50 ml) syringe. In an embodiment, this syringe can be a (50 ml) syringe with luer lock which is first connected to a vacutainer holder.

[0169] In a next step, said obtained plasma sample is transferred to one or more concentrator tubes having a concentrator column having a molecular weight cut-off filter membrane with a conical shape and a septum cap. The concentrator tubes can be placed inside tube holders which can be positioned opposite to each other in the centrifuge. The concentrator tubes can be centrifuged at 3400 RPM (2200 g) for 10 minutes. After centrifugation, the septum cap of the one or more concentrator tubes is disinfected, for instance with an alcohol wipe.

[0170] In an embodiment, the concentrated plasma sample can be aspirated from the concentrator tube(s) with a long needle into a (20 ml) syringe. In an embodiment, an adapter can be connected to the syringe for easy distribution to other syringes.

[0171] In a preferred embodiment, said a2-macroglobulin enriched sample comprises at least 0.20 mg / ml, more preferably at least 0.30 mg / ml, more preferably at least 0.40 mg / ml, more preferably at least 0.45 mg / ml, more preferably at least 0.50 mg / ml a2-macroglobulin.

[0172] In a preferred embodiment, said O2-macroglobulin enriched sample comprises at least 0.20 mg / ml, more preferably at least 0.30 mg / ml, more preferably at least 0.40 mg / ml, more preferably at least 0.45 mg / ml, more preferably at least 0.50 mg / ml a2-macroglobulin after said incubation step.

[0173] Said 02-macroglobulin protein concentration in said sample can be determined by any suitable technique known from the art for measuring a protein concentration, such as an immuno-enzymatic method, an immuno-histochemical method or mass spectrometry. In an embodiment, said 02-macroglobulin protein concentration in said sample is determined by means of ELISA (Enzyme Linked Immunosorbent Assay) or quantitative mass spectrometry (for instance LC-MS / MS shotgun proteomic analysis). In an embodiment, said a2-macroglobulin messenger RIMA (mRNA) concentration in said sample can be determined by any suitable technique known from the art for measuring a mRNA concentration, such as for instance reverse transcription polymerase chain reaction (RT-PCR).

[0174] In a preferred embodiment, the 02-macroglobulin concentration in said 02- macroglobulin enriched sample has a volume based fold increase of at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 11, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, such as 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 fold compared to a whole blood sample.

[0175] In a preferred embodiment, the a2-macroglobulin concentration in said 02- macroglobulin enriched sample has a volume based fold increase of at least 8, more preferably at least 9, more preferably at least 10, more preferably at least 11, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, such as 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 fold compared to a plasma sample.

[0176] The term "volume based fold increase" as used herein refers to the ratio between the concentration of Alpha-2-macroglobulin (A2MG) in a concentrated sample and the concentration of A2MG in the corresponding untreated plasma or whole blood sample (before concentration step), normalized to volume.

[0177] In a further aspect, the invention relates to a blood-derived product, wherein said blood-derived product is an 02-macroglobulin enriched sample comprising at least 0.20 mg / ml, more preferably at least 0.30 mg / ml, more preferably at least 0.40 mg / ml, more preferably at least 0.45 mg / ml, more preferably at least 0.50 mg / ml a2-macroglobulin obtainable by aforementioned method.

[0178] In an embodiment, said a?-r lacroglobulin enriched « ample comprises 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1.00, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.10, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.20, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.40, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2.00, 2.01, 2.02, 2.03, 2.04, 2.05, 2.06, 2.07, 2.08, 2.09, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, 2.23, 2.24, 2.25, 2.26, 2.27, 2.28, 2.29, 2.30, 2.31, 2.32, 2.33, 2.34, 2.35, 2.36, 2.37, 2.38, 2.39, 2.40, 2.41, 2.42, 2.43, 2.44, 2.45, 2.46, 2.47, 2.48, 2.49, 2.50 r ig / ml Q2-macroglobulin , >r any value in between.

[0179] In an embodiment, said a2-macroglobulin enriched sample comprises between 0.20- 0.25 mg / ml, between 0.25-0.30 mg / ml, between 0.30-0.35 mg / ml, between 0.35-

[0180] 0.40 mg / ml, between 0.40-0.45 mg / ml, between 0.45-0.50 mg / ml, between 0.50-

[0181] 0.55 mg / ml, between 0.55-0.60 mg / ml, between 0.60-0.65 mg / ml, between 0.65-

[0182] 0.70 mg / ml, between 0.70-0.75 mg / ml, between 0.75-0.80 mg / ml, between 0.80-

[0183] 0.85 mg / ml, between 0.85-0.90 mg / ml, between 0.90-0.95 mg / ml, between 0.95-

[0184] 1.00 mg / ml, between 1.00-1.05 mg / ml, between 1.05-1.10 mg / ml, between 1.10-

[0185] 1.15 mg / ml, between 1.15-1.20 mg / ml, between 1.20-1.25 mg / ml, between 1.25-

[0186] 1.30 mg / ml, between 1.30-1.35 mg / ml, between 1.35-1.40 mg / ml, between 1.40-

[0187] 1.45 mg / ml, between 1.45-1.50 mg / ml, between 1.50-1.55 mg / ml, between 1.55-

[0188] 1.60 mg / ml, between 1.60-1.65 mg / ml, between 1.65-1.70 mg / ml, between 1.70-

[0189] 1.75 mg / ml, between 1.75-1.80 mg / ml, between 1.80-1.85 mg / ml, between 1.85-

[0190] 1.90 mg / ml, between 1.90-1.95 mg / ml, between 1.95-2.00 mg / ml, between 2.00-

[0191] 2.05 mg / ml, between 2.05-2.10 mg / ml, between 2.10-2.15 mg / ml, between 2.15- 2.20 mg / ml, between 2.20-2.25 mg / ml, between 2.25-2.30 mg / ml, between 2.30- 2.35 mg / ml, between 2.35-2.40 mg / ml, between 2.40-2.45 mg / ml or between 2.45- 2.50 mg / ml a2-macroglobulin.

[0192] The inventors discovered that the method of the current invention, more specifically, a method wherein a collected whole blood sample is preferably first subjected to one or more centrifugation steps to obtain a plasma sample and wherein the obtained plasma sample is subsequently subjected to a filtration step using a molecular weight cut-off membrane of more than 100 kDa with a conical shape to obtain a concentrated plasma sample, allows to obtain an A2MG enriched sample. The inventors found that higher concentrations of A2MG can be obtained by using a concentrator column having a filter membrane with a conical shape.

[0193] In a preferred embodiment, said A2MG enriched sample further has an increased albumin, total protein, glucose and / or globulin concentration compared to a whole blood sample.

[0194] In a preferred embodiment, said A2MG enriched sample further has an increased albumin, total protein, glucose and / or globulin concentration compared to a plasma sample obtained after centrifugation of a collected whole blood sample.

[0195] In a preferred embodiment, said A2MG enriched sample further has an increased alkalin phosphatase, aspartate amino transferase and / or creatin phosphokinase concentration compared to a plasma sample obtained after centrifugation of a collected whole blood sample.

[0196] As described above, white blood cells may pose a concern in tissue regenerative efforts due to the pro-inflammatory mediators contained in white blood cells.

[0197] As such, in a preferred embodiment, said O2-macroglobulin enriched sample comprises less than 1%, preferably less than 0.5%, white blood cells. In a preferred embodiment, said O2-macroglobulin enriched sample comprises less than 500 white blood cells / pl. In a more preferred embodiment, said a2-macroglobulin enriched sample comprises less than 450, 400, 350, 300, 250, 200 or 100 white blood cells / pl. In a more preferred embodiment, said a2-macroglobulin enriched sample comprises less than 90, 80, 70, 60, 50, 40, 30 or 20 white blood cells / pl. In a preferred embodiment, said a2-macroglobulin enriched sample comprises less than 29% red blood cells. In a preferred embodiment, said O2-macroglobulin enriched sample comprises even less red blood cells, such as less than 25%, less than 20%, less than 15%, less than 10 %, less than 5% or less than 1% red blood cells. In a preferred embodiment, said O2-macroglobulin enriched sample comprises less than 30xl03red blood cells / pl. In a more preferred embodiment, said a2-macroglobulin enriched sample comprises less than 3xl03, lxlO3, 5xl02, 1 xlO2blood cells / pl. In a more preferred embodiment, said a2-macroglobulin enriched sample comprises less than 90, 80, 70, 60, 50, 40, 30, 20 or 10 red blood cells / pl.

[0198] In a preferred embodiment, the total protein content in said O2-macroglobulin enriched sample is between 10 g / L and 500 g / L, more preferably between 10 g / L and 100 g / L, such as between 50 and 60 g / L.

[0199] In a preferred embodiment, the variation in size of said red blood cells in said 02- macroglobulin enriched sample is minimal. In a preferred embodiment, said red cell distribution width of said red blood cells in said O2-macroglobulin enriched sample is less than 1%, more preferably less than 0.5%.

[0200] In a preferred embodiment, said O2-macroglobulin enriched sample comprises less than 1% granulocytes, more preferably less than 0.5% granulocytes.

[0201] In a further aspect, the invention relates to a concentrator tube for obtaining an a2- macroglobulin enriched sample, said concentrator tube comprising a concentrator column with a molecular weight cut-off filter membrane having a conical shape. In a preferred embodiment, said concentrator tube comprises a first cylinder and a second cylinder each defined by a cylindrical wall and a first and second end. In a preferred embodiment, said first cylinder comprises said concentrator column with a molecular weight cut-off filter membrane having a conical shape. In a preferred embodiment, said first end of said second cylinder is configured to be connected to the first end of the first cylinder and said second end of said second cylinder has a closed end portion for receiving the filtrate. In an preferred embodiment, said second cylinder has a height of more than 2 cm, more preferably more than 3 cm, more preferably more than 4cm or 5 cm.

[0202] In a preferred embodiment, said concentrator tube comprises a septum cap which is configured to be connected to the second end of the first cylinder. In an embodiment, said septum cap of said concentrator tube comprises more than one septum. These septa may be arranged concentrically, linearly, or in a polygonal configuration, depending on user needs. The septa may have different durometers to accommodate various needle gauges or automated pipette tips, with resealing capability after puncture to maintain a sterile barrier.

[0203] The use of a septum cap significantly enhances sterility by eliminating the need to remove the cap, which could expose the interior of the separator tube to airborne contaminants or accidental contact. The septa act as self-sealing membranes, allowing repeated puncture with minimal risk of microbial ingress. This design supports aseptic workflows.

[0204] In a further aspect, the invention relates to a kit comprising one or more concentrator tubes as described above and further comprising: one or more separator tubes, said one or more separator tubes comprising a clot activator; one or more syringes.

[0205] Said separator tubes are as described above. Preferably said one or more separator tubes are not glass tubes, but plastic tubes suited for centrifugation (for instance centrifugation tubes formed from a thermoplastic polymer, such as polypropylene or polycarbonate). Such plastic tubes facilitate white blood cell adherence. Said one or more separator tubes comprise a clot activator. Common clot activators include silica particles (silicon dioxide), which are typically coated on the interior walls of the tube to initiate the intrinsic coagulation pathway. In some configurations, the clot activator may consist of finely divided glass particles or microparticles that likewise promote clot formation upon contact with whole blood. Alternatively, thrombin may be employed as a clot activator to accelerate the coagulation process. The clot activator may be used alone or in combination with a gel separator, as is the case in serum separator tubes (SST), which combine silica-based activators with polymeric gel barriers to facilitate separation of serum following centrifugation.

[0206] In a preferred embodiment, said one or more separator tubes comprise a septum cap. The septum cap may be formed from elastomeric materials such as medicalgrade silicone, butyl rubber, or thermoplastic elastomers, optionally coated with a thin layer of fluoropolymer to reduce protein adhesion and improve chemical resistance. The cap may comprise a single septum, dual septa, or even multiple discrete septa integrated into a single cap body.

[0207] In a preferred embodiment, the septum cap comprises more than one septum, thereby enabling distinct pathways for sample introduction and sample extraction.

[0208] These septa may be arranged concentrically, linearly, or in a polygonal configuration, depending on user needs. The septa may have different durometers to accommodate various needle gauges or automated pipette tips, with resealing capability after puncture to maintain a sterile barrier.

[0209] In an embodiment, the kit comprises one or more syringes for blood collection, such as one or more 50 ml syringes with or without luer lock. In an embodiment, the kit comprises one or more syringes for plasma collection after centrifugation in the separator tubes, such as one or more 50 ml syringes with or without luer lock.

[0210] In an embodiment, the kit comprises one or more vacutainer holders. A vacutainer holder (also known as a needle holder or tube holder) is a medical device designed to securely connect a (Vacutainer) needle with a blood collection tube or a syringe, allowing for safe and efficient blood collection.

[0211] In an embodiment, the kit comprises one or more needles to aspirate the concentrated plasma from the concentrator tubes.

[0212] In an embodiment, the kit comprises one or more syringes for collecting the aspirated plasma from the concentrator tubes, for instance one or more 20 ml syringes.

[0213] In an embodiment, the kit comprises one or more adapters for transferring fluids (such as the concentrated plasma sample, the a2-macroglobulin enriched sample) from one syringe to another syringe.

[0214] In an embodiment, the kit comprises one or more vials with anticoagulant (for instance ACD-A anticoagulant).

[0215] In an embodiment, the kit comprises one or more alcohol wipes for disinfection.

[0216] As described above, given that A2MG is a binding protein of IGFBP-1 resulting in enhanced IGF effects, an O2-macroglobulin enriched sample can be used in the treatment of a musculoskeletal disease, preferably a bone disease, a tendon disease or a joint disease in a subject.

[0217] In a further aspect, the invention relates to aforementioned blood-derived product, for use in the treatment of a musculoskeletal disease, preferably a bone disease, a tendon disease or a joint disease in a subject. Alternatively or in addition, said musculoskeletal disease may affect tendons and / or ligaments. In an embodiment, the aforementioned blood-derived product is used in the treatment of synovitis, arthritis, bursitis, and / or tenosynovitis. In an embodiment, the aforementioned blood-derived product is used in the treatment of chronic tendinopathies, acute ligamentous injuries, muscle injuries and / or for intraoperative augmentation.

[0218] In an embodiment, the aforementioned blood-derived product is used in the treatment of epicondylitis, patellar tendonitis, plantar fasciitis and / or osteoarthritis.

[0219] In an embodiment, the aforementioned blood-derived product is used to augment and accelerate healing in Achilles tendon and rotator cuff repairs.

[0220] In an embodiment, the aforementioned blood-derived product is used to accelerate anterior cruciate ligament (ACL) graft / tunnel incorporation.

[0221] In an embodiment, said blood-derived product for use according to the current invention is configured for parenteral administration, preferably for intraosseous, periosseous, intraarticular, periarticular administration, or for intratendon, peritendon, intraligament, or periligament administration.

[0222] The blood-derived product for use according to current invention is preferably configured for parenteral administration. Parenteral administration has the advantage that a local injection can take place and the site of injury can be specifically targeted and treated. Said targeted treatment by the blood-derived product of the current invention is safe and efficient and does not involve complex functionality measurements like medicines for oral administration. Depending on the site of injury different locations of application routes are favored. A person skilled in the art is familiar with these application routes. The blood-derived product for use according to current invention is preferably administered by injection, using a needle and a syringe. In a particular embodiment, the blood-derived product is configured for intraosseous or periosseous administration. Intraosseous administration or delivery generally refers to a method whereby a treatment is delivered, directly or indirectly, into the bone (trabecular or cortical). Periosseous administration or delivery generally refers to a method whereby a treatment is delivered in the surroundings of a bone (especially around the fracture / damage site).

[0223] In another particular embodiment, the blood-derived product is configured for intraarticular or periarticular administration. Intraarticular administration or delivery generally refers to a method whereby a treatment is delivered, directly or indirectly, into the synovial capsule of an articulating joint. Periarticular administration or delivery generally refers to a method whereby a treatment is delivered in the surroundings of the synovial capsule of an articulating joint and / or the subchondral bone.

[0224] In another particular embodiment, the blood-derived product is configured for intratendon or peritendon administration. In another particular embodiment the blood-derived products are configured for intraligament or periligament administration.

[0225] Also intended is the use of the blood-derived product as described above for the manufacture of a medicament for the treatment of a musculoskeletal disease, preferably a bone disease, a tendon disease or a joint disease, more preferably osteoarthritis, or tendinopathies such as supraspinatus tendinopathy and Achilles tendon rupture.

[0226] Further intended is a method for treating a musculoskeletal disease, preferably a bone disease, a tendon disease or a joint disease (alternatively or in addition, said disease may affect tendons and / or ligaments), in a subject in need of such treatment, comprising administering to said subject a therapeutically or prophylactically effective amount of the blood-derived product as described above.

[0227] In an embodiment, the blood-derived product is an orthobiologic. Orthobiologics are organic and synthetic materials that help in the cure of musculo-skeletal problems and are utilized in orthopedic surgery, both in and out of the surgical theater, to augment the possibilities of curing bone and soft tissue lesions. In an embodiment, the current invention relates to a blood-derived product for use in the treatment of osteoarthritis. Osteoarthritis is a progressively worsening inflammation of the joint caused by the deterioration of cartilage. In a healthy joint, cartilage acts as a cushion to allow the joint to move smoothly through its full range of motion. In cases of osteoarthritis, this cartilage cushion begins to break down because of factors such as age, injury, repetitive stress, disease or genetic predisposition. The loss of this protective cushion results in pain, inflammation, decreased range of motion, and the development of bone spurs. Diagnosis is typically based on signs and symptoms, with medical imaging and other tests used to support or rule out other problems. Joint diseases are a major cause of lameness in horses and have considerable negative economic consequences resulting from the reduced active lifespan of the horses concerned.

[0228] In an embodiment, the current invention relates to a blood-derived product for use in the treatment of tendinopathies, such as supraspinatus tendinopathy and Achilles tendon rupture. Tendinopathy is a type of tendon disorder that results in pain, swelling, and impaired function. Supraspinatus tendinopathy is a term used to describe tears, calcifying tendinopathy, tendinosis and / or injuries in and around the tendon of the supraspinatus muscle, and is a cause of forelimb lameness. The supraspinatus is an important passive stabiliser of the shoulder joint, and is responsible for shoulder extension and advancing the limb. Injury to the tendon of the supraspinatus muscle causes inflammation. Tearing of the tendon fibers and the resulting inflammation can lead to mineralization and calcification of the tendon, which are a source of pain and lameness. The Achilles tendon's main function is rearlimb forward progression, and it contributes to passive support of the hock. The etiology of Achilles tendon injuries is usually traumatic. Depending on the trauma, the severity of the lesion may vary considerably, leading to stretching, small or partial lacerations or a complete rupture.

[0229] In a further aspect, the invention further relates to aforementioned blood-derived product for use in the treatment of a respiratory disease, such as asthma.

[0230] Such respiratory disease could be selected from a chronic respiratory disease, such as asthma, chronic obstructive pulmonary disease (COPD) (including chronic bronchitis, emphysema), bronchiectasis, cystic fibrosis, allergic rhinitis or chronic sinusitis. In an preferred embodiment, said aforementioned blood-derived product is used in the treatment of asthma.

[0231] When the disease to be treated is a respiratory disease, the aforementioned blood- derived product can be administered orally, intranasally, inhalationally, or via intratracheal instillation, depending on the severity and location of the condition. In a preferred embodiment, the product may be aerosolized and delivered using a nebulizer, allowing the active components to reach the lower respiratory tract directly, thereby enhancing local bioavailability and therapeutic efficacy. The use of a nebulizer is particularly advantageous for diseases affecting the bronchioles and alveolar regions, such as asthma, bronchitis, or pneumonia.

[0232] In an embodiment, more than one dose can be administered, for instance a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 doses at 48- to 72-hour intervals, or until clinical symptoms are adequately controlled. Additional cycles of multiple doses may be initiated if necessary. In an embodiment, following completion of the initial regimen, a second cycle could be initiated with a lower or higher dosage.

[0233] In a preferred embodiment, for treatment of asthma, 8 doses at 48- to 72-hour intervals can be administered.

[0234] In an embodiment, the aforementioned blood-derived product can be diluted with a solution, for instance with sterile physiological saline or Lactated Ringer's Solution (LRS) before administration.

[0235] In an embodiment, the aforementioned blood-derived product is twice diluted. For instance, each administration (each dosage) can consist of a volume of blood-derived product combined with an equal volume of dilution solution, for instance 4 mL of blood-derived product diluted with 4 mL of sterile physiological saline (0.9%) or Lactated Ringer's Solution (LRS), resulting in an 8 mL volume per administration. In an embodiment, the aforementioned blood-derived product is three times, four times or more times diluted.

[0236] In an embodiment, the aforementioned blood-derived product can be combined with other therapies to treat the disease, for instance horses currently receiving treatment for equine asthma may continue their existing therapy concurrently with application of the blood-derived product of the invention (for instance an a2-macroglobulin enriched sample).

[0237] In an embodiment, the aforementioned blood-derived product can be used in the acute and / or in the chronic inflammatory phase of the disease to be treated.

[0238] The blood-derived product may be administered as a single therapeutically effective dose or administered as multiple therapeutically effective doses for parenteral administration, according to the patient's physiopathological condition and the protocol established by the medicinal practitioner.

[0239] In an embodiment, the blood-derived product according to the current invention is intraarticular or locally administered in a single or multiple therapeutically effective dose(s). The dose can be adapted to the targeted joint and the body weight of the subject. Specifically in the case of tendinopathies, the blood-derived product according to the current invention is periarticular, peritendinous or intratendinous administered in a single or multiple therapeutically effective dose(s). The dose can be adapted to the targeted site and the body weight of the subject.

[0240] In an embodiment, the blood-derived product according to the current invention is administered orally, intranasally, inhalationally, or via intratracheal instillation.

[0241] In an embodiment, multiple therapeutically effective doses of the blood-derived product can be given several weeks apart or on an as needed basis.

[0242] In a preferred embodiment, the blood-derived product for use according to the present invention is formulated for administration to a subject by means of local injection or infusion.

[0243] In a preferred embodiment, the blood-derived product for use according to the present invention is formulated for administration to a subject by means of inhalation, for instance by means of a nebulizer.

[0244] In an embodiment, a single dose is administered. In an alternative embodiment, the blood-derived product is administered at least twice, at least three times, at least four times, at least five times, preferably with intervals. In another or further embodiment, the treatment further comprises: multiple administrations of the blood-derived product, for example multiple local administrations, preferably per equine, feline or canine patient, wherein said multiple doses are administered at various time points, including but not limited to one or more of the following time points 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 7 days (1 week) apart, 2 weeks apart, 3 weeks apart, 4 weeks apart, 5 weeks apart, 6 weeks apart, 7 weeks apart, 8 weeks apart, 3 months apart, 6 months, 9 months apart, and / or 1 year apart. Preferably each dose is administered at least 2 weeks apart, more preferably at least 3 weeks apart, even more preferably at least 4 weeks apart, and most preferably at least 6 weeks apart.

[0245] In an embodiment, one dosage of said blood-derived product has a volume of about 0.5 to 5.0 ml, such as of about 1.0 to 5.0 ml, about 0.5 to 3.0 ml, about 0.5 to 2.0 ml. In another or further embodiment, one dosage of said blood-derived product has a volume of maximally about 5 ml, preferably maximally about 4 ml, more preferably maximally about 3 ml, more preferably maximally about 2 ml. This amount is suitable for local administration.

[0246] In an embodiment, one dosage of said blood-derived product has a volume of about 0.5 to 10.0 ml, such as 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 ml. In an embodiment, one dosage of said blood- derived product has a volume between 0.5-2.0 ml, between 2.0-4.0 ml, between 4.0- 6.0 ml, between 6.0-8.0 ml or between 8.0-10.0 ml.

[0247] In an embodiment, the dosage given to the subject (for instance an equine) depends on the intended location for administration. In an embodiment, when the intended location for administration is the navicular bursa, the dosage is between 1.0-3.0 ml, such as 2.0 ml. In an embodiment, when the intended location for administration is the coffin, pastern or the fetlock, the dosage is between 3.0-5.0 ml, such as 4.0 ml. In an embodiment, when the intended location for administration is the elbow / shoulder, the dosage is between 4.0-6.0 ml. In an embodiment, when the intended location for administration are the lower hock joints, the dosage is between 1.0-3.0 ml, such as 2.0 ml. In an embodiment, when the intended location for administration are the talocrural joints, the dosage is between 3.0-5.0 ml, such as 4.0 ml. In an embodiment, when the intended location for administration is the stifle joint, the dosage is between 5.0-7.0 ml, such as 6.0 ml. In an embodiment, when the intended location for administration is the sacro-iliac joint, the dosage is between 6.0-10.0 ml. In an embodiment, when the intended location for administration are the cervical facet joints, the dosage is between 1.0-3.0 ml, such as 2.0 ml. In an embodiment, when the intended location for administration is intralesional in a tendon / ligament, the dosage is between 0.5 -3.0 ml, such as between 1.0-2.0 ml. In an embodiment, when the intended location for administration is perilesional in a tendon / ligament, the dosage is between 3.0-7.0 ml, such as between 4.0-6.0 ml.

[0248] Said dosage may be formulated in a vial or in a pre-filled syringe.

[0249] In an embodiment, the volume of the blood-derived product which is administered per injection to a patient is adapted in accordance with the patient's body weight. In another embodiment, a fixed volume is administered per patient.

[0250] The inventors have further discovered that a particularly effective treatment is achieved by a dosing regimen comprising at least two dosages of the blood-derived product for use as described above in any of the embodiments.

[0251] In an embodiment, said second dose is identical to the first dose. In another embodiment, said second dose is lower than the first dose. In yet another embodiment, said second dose is higher than the first dose.

[0252] In an embodiment, a third, fourth and / or even a fifth amount of said blood-derived product may be administered, preferably locally, to said patient.

[0253] The blood-derived product for use according to the current invention, possibly together with further components as described above, will by preference be frozen in order to allow long-time storage of the product. Preferably the blood-derived product will be frozen at low and constant temperature, such as a temperature below -20°C. These conditions allow a save storage of the blood-derived product, and enable the product to keep its biological characteristics.

[0254] In a more preferred embodiment, the blood-derived product according to the current invention can be stored for at least 6 months at a maximum temperature of -80°C, optionally in liquid nitrogen. Afterwards, the blood-derived product for use according to the current invention is preferably thawed before administration at a temperature around room temperature, preferably at a temperature between 20°C and 37°C, more preferably at a temperature between 25°C and 37°C, and in a time span of maximal 20 minutes, preferably maximal 10 minutes, more preferably maximal 5 minutes.

[0255] Said subject to which the blood-derived product is administered (the recipient), can be any mammal in need of treatment, including, but not limited to, humans, domestic and farm animals, zoo animals, sport animals, pet animals, companion animals and experimental animals, such as, for example, mice, rats, rabbits, dogs, cats, cows, horses, pigs and primates, e.g., monkeys and apes; especially horse, human, cat, dogs, rodents, etc.

[0256] In a preferred embodiment, said blood-derived product is used in the treatment of a musculoskeletal disease in an equine, a feline or a canine.

[0257] In an embodiment, said blood-derived product is used for autologous administration.

[0258] "Autologous" administration of a blood-derived product in the present context refers to a blood-derived product from a blood donor being administered to a recipient, wherein both recipient and donor are the same subject.

[0259] In an alternative embodiment, the use of allogeneic or xenogeneic blood is a more favorable option as they offer a stringent selection of healthy and high-quality donors. They allow the production of a ready-to-use product, avoiding collecting blood from each individual patient.

[0260] In an embodiment, said blood-derived product is used for allogeneic or xenogeneic administration.

[0261] "Allogeneic" administration of a blood-derived product in the present context refers to a blood-derived product from a blood donor being administered to a recipient, wherein both recipient and donor are of the same species, but are not the same.

[0262] "Xenogeneic" administration of MSCs in the present context refers to a blood-derived product from a blood donor being administered to a recipient, wherein the recipient and the donor are from different species. In particular, these donors will be tested on common current transmittable diseases or pathologies, in order to avoid the risk of horizontal transmission of these pathologies or diseases through the blood-derived product. Preferably, the donors / donor animals are kept in quarantine. When using donor horses they can be, for example tested for the following pathologies, viruses or parasites: equine infectious anemia (EIA), equine rhinopneumonitis (EHV-1, EHV-4), equine viral arteritis (EVA), West Nile virus (WNV), African horse Sickness (AHS), dourine (Trypanosoma), equine piroplasmosis, glanders (malleus, glanders), equine influenza, Lyme borreliosis (LB) (Borrelia burgdorferi, Lyme disease), Leptospirosis, L. canicola, L. autumnalis, L. Pomona, L. Bratislava, L. copenhageni (SG L. icterohaem.), L. tarassovi, L. sejroe, L. grippotyphosa, L. ballum, Piroplasmosis Th. Equi, Piroplasmosis B. caballi, A. phagocytophilum, Eq. Herpesvirus Type 1, Eq. Herpesvirus Type 4, Eq. Influenza A, Equ. Parvovirus, Bornavirus.

[0263] Because of the relatively low body size (and blood volume) of felines and canines compared to for example equines or humans, the use of xenogeneic (e.g. human or equine) blood is preferred above allogeneic feline or canine blood, especially for commercial applications, such as for use in the treatment of feline and canine musculoskeletal diseases.

[0264] The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention.

[0265] EXAMPLES AND / OR DESCRIPTION OF FIGURES

[0266] Example 1: Methods for obtaining a platelet rich plasma sample or an a2-macroqlobulin enriched sample.

[0267] Whole blood was obtained from six different donor horses and used for evaluating six different blood processing methods (T1-T6, see Figures 1A-E) :

[0268] Tl. Baseline= Plasma tube

[0269] T2. Proton= a2-macroglobulin enriched sample without incubation step

[0270] T3. Proton Inc = a2-macroglobulin enriched sample obtained by means of a kit according to the method of the current invention, including an incubation step at 37°C for lh

[0271] T4. PRP= platelet rich plasma preparation without incubation step T5. PRP Inc = platelet rich plasma preparation obtained by means of a kit according to the method of the current invention, including an incubation step at 37°C for lh T6. a2MG EQ = a2-macroglobulin enriched sample obtained by a competitor's kit and method

[0272] As evidenced from Fig ures 1A and IB, the a2-macroglobulin enriched sample obtained by the method of the current invention (T3, "Proton Inc", including an incubation step at 37°C for lh) obtained the highest level of a2-macroglobulin per kit and the highest volume based fold increase.

[0273] The method of the current invention for obtaining a PRP sample (T5, "PRP Inc", including an incubation step at 37°C for lh) results in the highest number of platelets (see Figure 1C). Furthermore, the method of the current invention for obtaining a PRP sample (T5, "PRP Inc", including an incubation step at 37°C for lh) results in the highest IGF-1 ("IGF-I") levels in the PRP groups and substantially higher levels of IGF-1 (concentration and fold increase compared to baseline) in the currently reported a2-macroglobulin enriched sample as compared to an a2-macroglobulin enriched sample obtained by a competitor's kit and method (see Figures 1D-E). In addition, the method and the kit of the current invention allow to obtain an a2- macroglobulin enriched sample having higher total protein levels (see Figure IF), with or without incubation step ("Proton" and "Proton Inc", respectively), as compared to an a2-macroglobulin enriched sample obtained by a competitor's kit and method ("a2MG EQ").

[0274] Example 2: Method for obtaining an TRAP enriched sample

[0275] An IRAP enriched sample is obtained by a method according to the current invention. For this, a whole blood sample is obtained from a healthy donor horse and collected in a citrate bag. The whole blood is subsequently incubated for 24 hours at a temperature of 37°C. This incubation step allows the blood cells to produce IRAP and release the intracellular protein into the blood sample. After said incubation step, the serum containing IRAP is separated from the blood cells by centrifugation. After said centrifugation step, the serum containing IRAP is further subjected to a filtration step.

[0276] The IRAP concentration in said sample ("Cytolease") is determined by means of ELISA and compared to samples incubated at different conditions (for 2 hours and 24 hours at 4°C and for 2 hours and 6 hours at 37°C). The concentration of IRAP in the Cytolease sample (obtained by the method of the current invention) was also compared to an IRAP sample obtained by a kit and method from a competitor (having a modified syringe comprising glass beads for incubating the whole blood sample) and a serum sample , which were also incubated for 24 hours at 37°C (see Figure 2). As evident from figures 2 and 3, the method of the current invention allows to enrich the IRAP level in the blood sample, allowing to obtain an IRAP enriched blood-derived product.

[0277] Example 3: protocol for obtaining a PRP sample using a kit

[0278] Introduction:

[0279] The PRP kit is intended for the processing of an autologous blood product for the treatment of joints and tendons. The kit consists of medical grade disposable items. All parts are packaged and delivered sterile.

[0280] User instructions :

[0281] Step 1: blood collection

[0282] • Fill 2x 50ml syringes with 5ml each of sterile ACD-A anti-coagulant.

[0283] • Prepare the horse's jugular vein for aseptic blood collection.

[0284] • Aspirate 35 ml of blood into both 50 ml syringes until they are filled to 40 ml.

[0285] • Rotate the syringes several times so that the blood is well mixed with the anticoagulant.

[0286] Step 2: centrifugation removing white blood cells (WBCs) and red blood cells (RBCs)

[0287] • Fill 2x 50ml tubes using a sterile needle with the collected blood in the 50ml syringe (one syringe per tube).

[0288] • Place 50ml tube holders opposite to each other in the centrifuge.

[0289] • Place the 2x 50ml tubes in these holders opposite to each other.

[0290] • Centrifuge at 200 G (1300 RPM) for 15 minutes.

[0291] • After centrifugation, disinfect the septum of the blood tubes with the supplied alcohol wipe.

[0292] Step 3: centrifugation removing remaining RBCs

[0293] • Aspirate the yellow plasma supernatans with a 50 ml syringe and sterile needle through the septum of the 50ml tube (to keep a closed system at all times). Make sure to stay at least 1cm above the buffy coat (WBC pellet above the RBCs).

[0294] • Bring half of the syringe in each of 2x 50ml tubes.

[0295] • Place the 2x 50ml tubes in the 50ml tube holders opposite to each other. • Centrifuge for a second time at 200 G (1300 RPM), but now for 5 minutes to remove the remaining WBCs and RBCs.

[0296] • After centrifugation, disinfect the septum of the blood tubes with the supplied alcohol wipe.

[0297] Step 4: ultracentrifugation for platelet enrichment of the plasma

[0298] • Aspirate the yellow plasma supernatans with a 50 ml syringe and sterile needle through the septum of the 50ml tube (to keep a closed system at all times). Make sure to stay at least 1cm above the buffy coat (WBC pellet above the RBCs).

[0299] • Bring half of the syringe in each of 2x 50ml tubes.

[0300] • Place the 2x 50ml tubes in the 50ml tube holders opposite to each other.

[0301] • Centrifuge for a third time at 1000 G (2900 RPM), but now for 10 minutes to remove the remaining WBCs and RBCs.

[0302] • After centrifugation, disinfect the septum of the blood tubes with the supplied alcohol wipe.

[0303] • Remove 2 / 3rd of the yellow plasma supernatans with a 50 ml syringe which contains the platelet poor plasma using a sterile needle through the septum of the 50ml tubes.

[0304] • The remaining l / 3rd of the yellow plasma and cell pellet contains the platelets that need to be resuspended with a long needle provided and can be aspirated in 5ml syringes. Incubate the platelet rich plasma (PRP) product for at least 30 minutes at a temperature between 28-41°C.

[0305] • Put a sterile needle on the 5ml syringe or a sterile cap as provided for further storage.

[0306] • In case a platelet lysate needs to be obtained, perform at least one freeze-thaw cycle in -20°C before injection.

[0307] Storage :

[0308] Use the plasma within 4 hours after preparation. Unused plasma can be stored frozen in sterile syringes with a sterile cap at -20°C for up to 12 months.

[0309] Example 4: protocol for obtaining an IRAP-enriched sample using a kit

[0310] User instructions :

[0311] Step 1: blood collection and incubation for white blood cell (WBC) stimulation

[0312] Prepare the horse's jugular vein for aseptic blood collection.

[0313] Fill the provided 6x sterile 10ml glass tubes using the vacutainer and needle. • Disinfect the septum of the blood tubes with the supplied alcohol wipe.

[0314] • Rotate the tubes several times so that the blood is well mixed with the glass components of the tubes.

[0315] • Incubate these 6 tubes at 37°C for 24 hours.

[0316] Step 2: centrifugation removing WBCs and red blood cells (RBCs)

[0317] • Place the 6x 10ml tubes balanced in a centrifuge (opposite to each other) after the 24 hour incubation period.

[0318] • Centrifuge at 1400 G (3400 RPM) for 10 minutes.

[0319] • After centrifugation, disinfect the septum of the blood tubes with the supplied alcohol wipe.

[0320] • Aspirate the yellow plasma supernatans from each 10ml tube with a sterile 5 ml syringe and sterile needle through the septum of the 50ml tube (to keep a closed system at all times). Make sure to stay at least 1cm above the buffy coat (WBC pellet above the RBCs).

[0321] • Put a sterile needle on the 5ml syringe or a sterile cap as provided for further storage.

[0322] Storage :

[0323] Use the plasma within 4 hours after preparation. Unused plasma can be stored frozen in sterile syringes with a sterile cap at -20°C for up to 12 months.

[0324] Example 5: protocol for obtaining an A2MG enriched sample using a kit

[0325] User instructions :

[0326] Step 1: blood collection

[0327] • Fill 2x 50ml syringes without luer lock with 5ml each of sterile ACD-A anticoagulant

[0328] • Prepare the horse's jugular vein for aseptic blood collection

[0329] • Aspirate 35 ml of blood into both 50 ml syringes until they are filled to 40 ml

[0330] • Rotate the syringes several times so that the blood is well mixed with the anticoagulant

[0331] Step 2: centrifugation

[0332] • Fill all blood tubes with the collected blood

[0333] • Place the red blood tube holders opposite to each other in the centrifuge

[0334] • Place the blood tubes in these holders.

[0335] • Centrifuge at 1400 G (3400 RPM) for 10 minutes.

[0336] • After centrifugation, disinfect the septum of the blood tubes with the supplied alcohol wipe. Connect the vacutainer holder to the 50 ml syringe with luer lock

[0337] Aspirate all plasma from the blood tubes into this 50 ml syringe

[0338] Step 3: centrifugation of the plasma

[0339] • Carefully divide the plasma into the supplied 2 tubes for ultracentrifugation.

[0340] • Place the blue tube holders opposite to each other in the centrifuge

[0341] • Place the filled tubes in these holders.

[0342] • Centrifuge at 1400 G (3400 RPM) for 10 minutes.

[0343] • After centrifugation, disinfect the septum of the blood tubes with the supplied alcohol wipe.

[0344] • Aspirate the plasma from each tube with a long needle into a 20 ml syringe.

[0345] • Connect the red adapter to the syringe for easy distribution to other syringes.

[0346] • Optionally, incubate the fractionated sample for at least 30 minutes at a temperature between 28-41°C.

[0347] Storage :

[0348] Use the plasma within 4 hours after preparation. Unused plasma can be stored frozen in sterile syringes with a sterile cap at -20°C for up to 12 months.

[0349] Example 6: Blood parameter analysis of an A2MG enriched sample obtained by a method and a concentrator tube according to an embodiment of the current invention.

[0350] The current invention allows to retrieve an A2MG enriched sample having at least 0.2 mg / ml a2-macroglobulin or wherein said a2-macroglobulin concentration in said a2- macroglobulin enriched sample has a volume based fold increase of at least 8 fold compared to a plasma sample. Furthermore, as depicted in table 1 below, the A2MG enriched sample comprises less than 50 white blood cells / pl, less than 50 red blood cells / pl and a total protein content between 10 and 500 g / L. A concentrator tube according to an embodiment of the current invention is depicted in Figure 4. Said concentrator tube has a first cylinder 1 having a first and a second end and a second cylinder 4 having a first and a second end. The first cylinder 1 comprises a concentrator column with a molecular weight cut-off filter membrane having a conical shape 2. The first end of said second cylinder 4 is configured to be connected to the first end of the first cylinder 1 and said second end of said second cylinder 4 has a closed end portion for receiving the filtrate 3. The septum cap is configured to be connected to the second end of the first cylinder 1. The septum cap 5 may comprise more than one septum, for example a centrally located septum 7 and a plurality of septa 6 arranged concentrically around the centrally located septum, as illustrated in Figure 5.

[0351] Parameter Result Lower Limit Upper Limit

[0352] Red blood cell count 0.00 0 x 106 / pL 0.03 x 106 / pL

[0353] White blood cell count 10 0 / pL 500 / pL

[0354] Total protein 53 10 g / L 500 g / L

[0355] Albumin 29.0 5 g / L 120 g / L

[0356] Color inspection Yellow Yellow Yellow

[0357] Label inspection Complete & correct Complete & correct Complete & correct

[0358] Infectious disease panel Absent Absent Absent

[0359] Aerobic bacteria Absent Absent Absent

[0360] Anaerobic bacteria Absent Absent Absent

[0361] Yeast Absent Absent Absent

[0362] Table 1

[0363] Embodiments

[0364] 1. A method for obtaining a blood-derived product, wherein said blood-derived product is chosen from platelet rich plasma (PRP), an a2-macroglobulin enriched sample or an Interleukin-1 receptor antagonist (IRAP) enriched sample, said method comprising: a. withdrawing blood from a blood donor and collecting said blood in a recipient comprising an anticoagulant, thereby obtaining a whole blood sample; b. optionally performing a fractionation step on said whole blood sample, thereby obtaining a fractionated blood sample; c. performing an incubation step on said whole blood sample or said fractionated blood sample, wherein said incubation step is performed for at least 30 minutes at a temperature between 28-41°C; d. optionally performing a fractionation step on said incubated sample. 2. Method according to embodiment 1, wherein said incubation step is performed for a period between 30 minutes and 4 hours.

[0365] 3. Method according to embodiment 1, wherein said incubation step is performed for more than 4 hours, such as 24 hours.

[0366] 4. Method according to any of the previous embodiments, wherein said blood donor is an equine.

[0367] 5. Method according to any of the previous embodiments 1-4, wherein an incubation step is performed on said fractionated blood sample.

[0368] 6. Method according to embodiment 5, wherein said fractionated blood sample is obtained by one or more centrifugation steps at a centrifugal force between 100 and 1000 g, thereby obtaining a platelet rich plasma sample comprising at least 50X109 platelets / liter after said incubation step.

[0369] 7. Method according to embodiment 5, wherein said fractionated blood sample is a concentrated plasma sample obtained by filtration using a molecular weight cutoff membrane of more than 100 kDa, thereby obtaining an a2-macroglobulin enriched sample comprising at least 0.2 mg / ml a2-macroglobulin after said incubation step.

[0370] 8. Method according to any of the previous embodiments 1-4, wherein an incubation step is performed on said whole blood sample and an Interleukin-1 receptor antagonist (IRAP) enriched sample comprising at least 10 ng / ml Interleukin- 1 receptor antagonist (IRAP) is obtained.

[0371] 9. Method according to embodiment 8, further comprising a fractionation step on said incubated sample.

[0372] 10. A blood-derived product obtainable by a method according to any of the previous embodiments 1-9, wherein said blood-derived product is: an Interleukin-1 receptor antagonist (IRAP) enriched sample comprising at least 10 ng / ml Interleukin-1 receptor antagonist (IRAP); a platelet rich plasma (PRP) sample comprising at least 50X109 platelets / liter and at least 50 ng / ml insulin-like growth factor (IGF)-I; or an a2-macroglobulin enriched sample comprising at least 0.2 mg / ml a2-macroglobulin.

[0373] 11. Blood-derived product according to embodiment 10, wherein said blood- derived product comprises less than 1% white blood cells.

[0374] 12. Blood-derived product according to any of the previous embodiment 10-11, for use in the treatment of a musculoskeletal disease, preferably a bone disease, a tendon disease or a joint disease in a subject. 13. Blood-derived product for use according to embodiment 12, wherein said subject is an equine, a feline or a canine.

Claims

CLAIMS1. A method for obtaining a blood-derived product, wherein said blood-derived product is an a2-macroglobulin enriched sample, said method comprising: withdrawing blood from a blood donor and collecting said blood in a recipient comprising an anticoagulant, thereby obtaining a whole blood sample; performing a fractionation step on said whole blood sample, thereby obtaining a fractionated blood sample; said fractionation step comprising:- subjecting said collected whole blood sample to one or more separation steps to obtain a plasma sample;- transferring said obtained plasma sample to one or more concentrator tubes, wherein said concentrator tube comprises a concentrator column having a molecular weight cut-off filter membrane with a conical shape;- performing a filtration step on said obtained plasma sample in said one or more concentrator tubes, wherein said filtration step involves centrifugation to obtain a concentrated plasma sample.

2. Method according to claim 1, wherein said filtration step uses a molecular weight cut-off filter membrane of more than 100 kDa.

3. Method according to claim 1, wherein said filtration step uses a molecular weight cut-off membrane of more than 50 kDa.

4. Method according to any of the previous claims, wherein said one or more separation steps comprise subjecting said collected whole blood sample to gravitational sedimentation and / or membrane filtration to obtain a plasma sample.

5. Method according to any of the previous claims, wherein said one or more separation steps comprise subjecting said collected whole blood sample to one or more centrifugation steps to obtain a plasma sample.

6. Method according to any of the previous claims, wherein said whole blood sample is transferred to one or more separator tubes prior to said one or more separation steps.

7. Method according to claim 6, wherein said one or more separator tubes and / or said one or more concentrator tubes comprise a septum cap.

8. Method according to claim 7, wherein said septum cap comprises more than one septum.

9. Method according to any of the previous claims 6-8, wherein said one or more separator tubes comprises a clot activator.

10. Method according to any of the previous claims 5-9, wherein said collected whole blood sample is subjected to one or more centrifugation steps at a centrifugal force between 1500 and 2500 g for 10 minutes to obtain a plasma sample.

11. Method according to any of the previous claims, wherein centrifugation of said concentrator tube occurs at a centrifugal force between 1500 and 2500 g for 8- 12 minutes, such as 10 minutes, to obtain a concentrated plasma sample.

12. Method according to any of the previous claims, wherein said fractionated blood sample is incubated for at least 30 minutes at a temperature between 28-41°C.

13. Method according to any of the previous claims, wherein said blood donor is an equine.

14. A blood-derived product obtainable by a method according to any of the previous claims 1-13, wherein said blood-derived product is an a2-macroglobulin enriched sample comprising at least 0.2 mg / ml a2-macroglobulin.

15. Blood-derived product according to claim 14, wherein the a2-macroglobulin concentration in said a2-macroglobulin enriched sample has a volume based fold increase of at least 8 fold compared to a plasma sample.

16. Blood-derived product according to any of the previous claims 14-15, wherein said blood-derived product comprises less than 50 white blood cells / pl.

17. Blood-derived product according to any of the previous claims 14-16, wherein said blood-derived product comprises less than 50 red blood cells / pl.

18. Blood-derived product according to any of the previous claims 14-17, wherein the total protein content in said blood-derived product is comprised between 10 and 500 g / L.

19. Blood-derived product according to any of the previous claims 14-18, for use in the treatment of a musculoskeletal disease, preferably a bone disease, a tendon disease or a joint disease in a subject.

20. Blood-derived product according to any of the previous claims 14-18, for use in the treatment of a respiratory disease in a subject.

21. Blood-derived product for use according to any of the previous claims 19-20, wherein said subject is an equine, a feline or a canine.

22. A concentrator tube for obtaining an a2-macroglobulin enriched sample, said concentrator tube comprising:- a first cylinder having a first end and a second end, said first cylinder comprising a concentrator column, wherein said concentrator column has a molecular weight cut-off filter membrane with a conical shape;- a second cylinder having a first end and a second end, wherein said first end of said second cylinder is configured to be connected to the first end of the first cylinder and wherein said second end of said second cylinder has a closed end portion; - a septum cap which is configured to be connected to the second end of the first cylinder.

23. Concentrator tube according to claim 22, wherein said septum cap has more than one septum.

24. A kit comprising one or more concentrator tubes according to any of the previous claims 22-23 and further comprising:One or more separator tubes, said one or more separator tubes comprising a clot activator;One or more syringes.

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