Clot types, kits and methods for preparing clots

Abstract

The present disclosure provides a method, clot type and kit for preparing a clot with a desired shape and volume. The clot is prepared from whole blood that has been drawn from a subject. To obtain a clot with optimal parameters, such as stability, moisture, homogeneity, i.e. the texture of the clot is substantially similar in every part, the whole blood is mixed with a clot initiator with specific properties, i.e. a coagulant or anti-anticoagulant, in a specific amount. This is achieved, among other things, by selecting the particle size of one of the clot initiators so that it dissolves faster in the blood and / or improves the suspension of the particles in the blood when the blood is still in a liquid state. Another parameter for obtaining the desired clot of the present invention is the selected amount of the clot initiator material mixed with the whole blood. Controlling these parameters results in an optimal clot that can be used in the treatment of the human body, for example, for the treatment of skin lesions, internal injuries (anal fistula, ostomy cavity, damaged tendons, etc.).

Description

[Technical field]

[0001] The present disclosure relates to the field of preparation of medical procedures, and in particular, preparation of blood clots for use in treating a subject. [Background technology]

[0002] References believed to be relevant to the subject matter of this disclosure are listed below for background. - US 9,180,142 - US 2020 / 0281775 - WO 2019 / 150355

[0003] Acknowledgement of the above references herein should not be construed as implying that they are in any way relevant to the patentability of the subject matter of the present disclosure.

[0004] The present disclosure provides a method, clot type and kit for preparing a clot, i.e. a clot with a desired shape and volume. The clot is prepared from whole blood that has been drawn from a subject. To obtain a clot with optimal parameters, such as stability, moisture, homogeneity, i.e. the texture of the clot is substantially similar in every part, the whole blood is mixed with a clot initiator, i.e. a coagulant or anti-anticoagulant, with specific properties in a specific amount. This is achieved, among other things, by selecting the particle size of one of the clot initiators so that it dissolves faster in the blood and / or improves the suspension of the particles in the blood when the blood is still in a liquid state. Another parameter is the selected amount of the clot initiator material mixed with the whole blood to obtain the desired clot of the present invention. By controlling these parameters, an optimal clot is obtained that can be used in the treatment of the human body, for example, for the treatment of skin lesions, internal injuries (anal fistula, ostomy cavity, damaged tendons, etc.).

[0005] Thus, a first aspect of the disclosure provides a method for preparing a blood clot, i.e., a volumetric blood clot, for use in treating a lesion or injury in a subject. The treatment can be external to the body, such as a skin lesion, or internal to the body, such as a fistula cavity or abdominal cavity. The method includes mixing a quantity of whole blood with calcium gluconate powder and kaolin powder, and allowing the whole blood to clot and form a clot. The term "clotting" refers to maintaining the mixture of whole blood with calcium gluconate powder and kaolin powder for a selected period of time until the blood has partially clotted and is in a flowable form. The period of time can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 15 minutes or more until the whole blood has clotted to a desired degree. The method is further characterized by at least one of the following: (i) the calcium gluconate powder has a particle size distribution D50 of 50μ to 200μ; (ii) the mixing comprises mixing 0.6 mg to 3.1 mg of kaolin powder per ml of whole blood; (iii) the mixing comprises mixing 2.5 mg to 8.1 mg of calcium gluconate powder per ml of whole blood; or (iv) the kaolin powder has a particle size distribution D50 of less than 20μ.

[0006] A particle size distribution DX means that X% of the particles in the powder are smaller than the nominal size associated with that distribution. For example, if the particle size distribution D50 is 125μ, then 50% of the particles will have a volume size of 125μ or less. By maintaining only particles of relatively small size, the resulting clot will be homogenous, i.e., the volumetric hardness of the clot will be substantially equal. This is due to the duration that particles of such size can remain suspended in the mixed whole blood. Larger particles will tend to sink in the blood and the resulting clot will not be homogenous.

[0007] It should be noted that for any aspect of the present disclosure, any combination of the described embodiments is applicable, in other words, any aspect of the present disclosure can be defined by any combination of the described embodiments.

[0008] In some embodiments of the method, the calcium gluconate powder has a particle size distribution D70 of 50μ to 200μ.

[0009] To prepare a clot with medical properties, the calcium gluconate powder is first crushed to a size of 2μ. The particles obtained by crushing usually undergo some granulation, so before using the powder to form a clot, the calcium gluconate powder is sieved to 120μ-125μ particles. Particle sizes below 120μ-125μ dissolve very quickly, so when used to form a clot, a more stable, leak-free clot is obtained.

[0010] In some embodiments of the method, the calcium gluconate powder has a particle size distribution D90 of 50μ to 200μ.

[0011] In some embodiments of the method, the calcium gluconate powder has a particle size distribution D90, D80, D70, D60, or D50 of about 125μ.

[0012] The term "about" should be understood as a deviation of up to 20% from the nominal value, for example, if the nominal value is about 125μ, this value should be considered to be between 100μ and 150μ.

[0013] In some embodiments of the method, the calcium gluconate powder has a particle size distribution D50, D60, D70, D80, D90, or D95 of 50μ to 200μ, 60μ to 190μ, 70μ to 180μ, 80μ to 170μ, 90μ to 160μ, 100μ to 150μ, 110μ to 140μ, or 120μ to 130μ.

[0014] In some embodiments of the method, the kaolin powder has a particle size distribution D99 of less than 50μ.

[0015] In some embodiments of the method, the kaolin powder has a particle size distribution D99 of less than 50μ.

[0016] The use of small particles of kaolin in preparing the clot results in a substantially homogenous clot because the kaolin particles remain suspended within the formed clot for a longer duration (when blood is at least partially in a liquid flowable form) and do not quickly sink to the bottom of the formed clot.

[0017] In some embodiments of the method, the kaolin powder has a particle size distribution D80 of less than 25μ.

[0018] In some embodiments of the method, the kaolin powder has a particle size distribution D90 of less than 3μ or between 1μ and 3μ.

[0019] In some embodiments of the method, the calcium gluconate powder has a particle size distribution of any of D50, D60, D70, D80, D90, or D95 of less than any of 30μ, 25μ, 20μ, 15μ, 10μ, 5μ, 3μ, 2μ, 1μ, or 0.1μ.

[0020] In some embodiments, the method comprises drawing whole blood from a subject, such that the clot is prepared from autologous whole blood.

[0021] In some embodiments, the method includes introducing an anticoagulant-containing liquid into said volume of whole blood prior to said mixing, i.e., after whole blood is drawn, an anticoagulant is added thereto to prevent undesirable blood clotting.

[0022] In some embodiments of the method, the introducing comprises introducing between 0.04 ml and 0.095 ml of anticoagulant-containing fluid per ml of whole blood.

[0023] In some embodiments of the method, the introducing comprises introducing about 0.07 ml of anticoagulant-containing fluid per ml of whole blood.

[0024] In some embodiments of the method, the mixing is performed within a clot mold, the clot mold having a desired shape that determines the shape of the clot prepared by the method.

[0025] In some embodiments of the method, the mixing comprises mixing 0.6 mg to 3.1 mg of kaolin powder with every 1 ml of whole blood.

[0026] In some embodiments of the method, the mixing comprises mixing about 1.75 mg of kaolin powder with every 1 ml of whole blood.

[0027] In some embodiments of the method, the mixing comprises mixing 0.8 mg to 2.9 mg, 1 mg to 2.7 mg, 1.2 mg to 2.5 mg, 1.4 mg to 2.3 mg, 1.6 mg to 2.1 mg, or 1.8 mg to 2 mg of kaolin powder with every ml of whole blood.

[0028] In some embodiments of the method, the mixing comprises mixing 2.5 mg to 8.1 mg of calcium gluconate powder with 1 ml of whole blood.

[0029] In some embodiments of the method, the mixing comprises mixing about 5.3 mg of calcium gluconate powder with every 1 ml of whole blood.

[0030] In some embodiments of the method, the mixing comprises mixing 2.9 mg to 7.7 mg, 3.3 mg to 7.3 mg, 3.7 mg to 6.9 mg, 4.1 mg to 6.5 mg, 4.5 mg to 6.1 mg, or 4.9 mg to 5.7 mg of calcium gluconate powder with every ml of whole blood.

[0031] Yet another aspect of the present disclosure provides a clotting mold including a clotting space defined by an upwardly facing walled base and configured to receive a selected volume of whole blood, the clotting space including calcium gluconate powder and kaolin powder, the clotting mold including at least one of the following: (i) the calcium gluconate powder having a particle size distribution D50 of 50μ to 200μ, (ii) 0.6mg to 3.1mg of kaolin powder per ml of the selected volume of whole blood, (iii) 2.5mg to 8.1mg of calcium gluconate per ml of the selected volume of whole blood, or (iv) the kaolin powder having a particle size distribution D50 of less than 10μ.

[0032] In some embodiments of the coagulation type, the calcium gluconate powder has a particle size distribution D70 of 50μ to 200μ.

[0033] In some embodiments of the coagulation type, the calcium gluconate powder has a particle size distribution D90 of 500μ to 200μ.

[0034] In some embodiments of the clot type, the calcium gluconate powder has a particle size distribution D90, D80, D70, D60, or D50 of about 125μ.

[0035] In some embodiments of the coagulation type, the particle size distribution of the calcium gluconate powder is any of 50μ to 200μ, 60μ to 190μ, 70μ to 180μ, 80μ to 170μ, 90μ to 160μ, 100μ to 150μ, 110μ to 140μ, or 120μ to 130μ.

[0036] In some embodiments of the clot type, the particle size distribution D99 of the kaolin powder is less than 50μ.

[0037] In some embodiments of the clot type, the particle size distribution D90 of the kaolin powder is less than 20μ or less than 10μ.

[0038] In some embodiments of the clot type, the particle size distribution D50 of the kaolin powder is less than 3μ or between 1μ and 3μ.

[0039] In some embodiments of the clot type, the calcium gluconate powder has a particle size distribution of any of D50, D60, D70, D80, D90, or D95 that is less than any of 30μ, 25μ, 20μ, 15μ, 10μ, 5μ, 3μ, 2μ, 1μ, or 0.1μ.

[0040] In some embodiments of the clot type, the whole blood intended to be contained comprises between 0.04 ml and 0.095 ml of anticoagulant-containing liquid per ml of whole blood, or in some embodiments, about 0.07 ml of anticoagulant-containing liquid per ml of whole blood.

[0041] In some embodiments, the clot form comprises between 0.6 mg and 3.1 mg of kaolin powder per ml of the selected amount of whole blood.

[0042] In some embodiments, the clot form comprises about 1.75 mg of kaolin powder per ml of the selected amount of whole blood.

[0043] In some embodiments, the clot form comprises between 0.8 mg and 2.9 mg, 1 mg and 2.7 mg, 1.2 mg and 2.5 mg, 1.4 mg and 2.3 mg, 1.6 mg and 2.1 mg, or 1.8 mg and 2 mg of kaolin powder per ml of whole blood.

[0044] In some embodiments, the clot form comprises between 2.5 mg and 8.1 mg of calcium gluconate powder per ml of the selected amount of whole blood.

[0045] In some embodiments, the clot form comprises about 5.3 mg of calcium gluconate powder per ml of the selected amount of whole blood.

[0046] In some embodiments, the clot form comprises between 2.9 mg and 7.7 mg, 3.3 mg and 7.3 mg, 3.7 mg and 6.9 mg, 4.1 mg and 6.5 mg, 4.5 mg and 6.1 mg, or 4.9 mg and 5.7 mg of calcium gluconate powder per ml of whole blood.

[0047] In some clot type embodiments, the volume of the clot space is sufficient to accommodate between 13 ml and 19 ml of whole blood.

[0048] In some clot type embodiments, the volume of the clot space is sufficient to accommodate about 16 ml of whole blood.

[0049] Yet another aspect of the present disclosure provides a kit for preparing a blood clot comprising calcium gluconate powder, kaolin powder, and a clot mold for mixing a selected amount of blood with the calcium gluconate powder and kaolin powder, characterized in that the calcium gluconate powder and kaolin powder have at least one of the following characteristics: (i) the calcium gluconate powder has a particle size distribution D50 of 50μ to 200μ, (ii) the kaolin powder has an amount of 0.6mg to 3.1mg per ml of the selected amount of whole blood, (iii) the calcium gluconate has an amount of 2.5mg to 8.1mg per ml of the selected amount of whole blood, or (iv) the kaolin powder has a particle size distribution D50 of less than 10μ.

[0050] In some embodiments, the kit includes equipment for collecting and containing whole blood.

[0051] In some embodiments, the kit comprises between 0.04 ml and 0.095 ml of an anticoagulant-containing fluid per ml of the selected amount of whole blood, which can be included separately within the kit or within the device to accommodate the collected whole blood.

[0052] In some embodiments of the kit, the selected volume is between 13 ml and 19 ml.

[0053] In some embodiments of the kit, the calcium gluconate powder has a particle size distribution D70 of 50μ to 200μ.

[0054] In some embodiments of the kit, the calcium gluconate powder has a particle size distribution D90 of 50μ to 200μ.

[0055] In some embodiments of the kit, the calcium gluconate powder has a particle size distribution D90, D80, D70, D60, or D50 of about 125μ.

[0056] In some embodiments of the kit, the calcium gluconate powder has a particle size distribution D50, D60, D70, D80, D90, or D95 of 50μ to 200μ, 60μ to 190μ, 70μ to 180μ, 80μ to 170μ, 90μ to 160μ, 100μ to 150μ, 110μ to 140μ, or 120μ to 130μ.

[0057] In some embodiments of the kit, the kaolin powder has a particle size distribution D99 of less than 50μ.

[0058] In some embodiments of the kit, the kaolin powder has a particle size distribution D80 of less than 10μ.

[0059] In some embodiments of the kit, the kaolin powder has a particle size distribution D50 of less than 3μ or between 1μ and 3μ.

[0060] In some embodiments of the kit, the calcium gluconate powder has a particle size distribution of any of D50, D60, D70, D80, D90, or D95 of less than any of 30μ, 25μ, 20μ, 15μ, 10μ, 5μ, 3μ, 2μ, 1μ, or 0.1μ.

[0061] In some embodiments, the kit comprises between 0.6 mg and 3.1 mg of kaolin powder per ml of the selected amount of whole blood.

[0062] In some embodiments, the kit comprises about 1.75 mg of kaolin powder per ml of the selected amount of whole blood.

[0063] In some embodiments, the kit comprises 0.8 mg to 2.9 mg, 1 mg to 2.7 mg, 1.2 mg to 2.5 mg, 1.4 mg to 2.3 mg, 1.6 mg to 2.1 mg, or 1.8 mg to 2 mg of kaolin powder per ml of whole blood.

[0064] In some embodiments, the kit comprises between 2.5 mg and 8.1 mg of calcium gluconate powder per ml of the selected amount of whole blood.

[0065] In some embodiments, the kit comprises about 5.3 mg of calcium gluconate powder per ml of the selected amount of whole blood.

[0066] In some embodiments, the kit contains between 2.9 mg and 7.7 mg, 3.3 mg and 7.3 mg, 3.7 mg and 6.9 mg, 4.1 mg and 6.5 mg, 4.5 mg and 6.1 mg, or 4.9 mg and 5.7 mg of calcium gluconate powder per ml of whole blood.

[0067] In some embodiments, the kit comprises a clot form of any one of the above embodiments or any combination thereof, wherein the kaolin powder and calcium gluconate powder are included within the clot form. [Brief description of the drawings]

[0068] In order to better understand the subject matter disclosed herein and to illustrate how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings in which:

[0069] 1A-1B are photographs of the results of particle size testing of kaolin powder. FIG. 1A shows the results at time zero, and FIG. 1B shows the results at time 15 minutes.

[0070] Below are example tests on various parameters of the powders used in the method for forming a clot. These tests are provided to illustrate the inventive embodiments and implementations of the present disclosure.

[0071] Testing of Calcium Gluconate (CG) Powder success criteria : An optimal clot is defined as: · Clotting time – A good clotting time is less than 6 minutes. Visual clot structural strength - Good structural strength is defined as the clot not crumbling as the user extracts and handles it. · CG Residue - visual assessment of the white powdery residue that accumulates at the bottom of the clot - 0 = no residue, 5 = 100% of the surface covered by residue.

[0072] method : Standard amounts of ACD-A anticoagulant and kaolin powder per ml of whole blood were used. 2 ml of ACD-A for 15 ml of blood = 0.133 ml of ACD-A / 1 ml of blood 28 mg kaolin powder per 15 ml blood = 1.87 mg kaolin / ml blood Different amounts of CG were tested. The CG particle size after sieving is 125 microns. 20 mg per 15 ml of blood = 1.33 mg CG / ml of blood 50 mg per 15 ml of blood = 3.33 mg CG / ml of blood 85 mg per 15 ml of blood = 5.66 mg CG / ml of blood 1 lO mg for 15 ml of blood = 7.33 mg CG / ml of blood 140 mg per 15 ml of blood = 9.33 mg CG / ml of blood The clotting time and the strength of the clot structure were recorded. Blood samples from five different volunteers were used.

[0073] result : The following table lists the results of this study: Table 1 JPEG2025504710000001.jpg238136JPEG2025504710000002.jpg37134

[0074] Kaolin powder testing success criteria : An optimal clot is defined as: 1. Clotting time--Good clotting time is less than 6 minutes. 2. Visual clot structural strength - Good structural strength is defined as the clot not crumbling as the user extracts and handles it.

[0075] method : Standard amounts of ACD-A anticoagulant and calcium gluconate powder per ml of whole blood were used. 2 ml of ACD-A per 15 ml of blood = 0.133 ml of ACD-A / ml of blood 85 mg CG powder per 15 ml of blood = 5.66 mg CG / ml of blood Different amounts of kaolin were tested. The kaolin particle size ranged from 1 to 3 microns. 5 mg per 15 ml of blood = 1.33 mg CG / ml of blood 10 mg per 15 ml of blood = 3.33 mg CG / ml of blood 20 mg per 15 ml of blood = 5.66 mg CG / ml of blood 30 mg per 15 ml of blood = 7.33 mg CG / ml of blood 40 mg per 15 ml of blood = 9.33 mg CG / ml of blood The clotting time and the strength of the clot structure were recorded. Blood samples from five different volunteers were used.

[0076] result : The following table lists the results of this study: Table 2 JPEG2025504710000003.jpg237140JPEG2025504710000004.jpg37140

[0077] Kaolin powder particle size test the purpose The purpose of this test is to establish an effective threshold at which kaolin powder will remain suspended in water for a sufficient period of time to simultaneously clot the entire volume of blood.

[0078] background When kaolin or other coagulation initiators (e.g., glass) come into contact with factor XI, blood will rapidly clot via the extrinsic pathway. The longer the kaolin particles are in contact with blood, the greater the likelihood of contact. Kaolin is a clay mineral that normally sinks in water due to its weight. If the kaolin particles could float freely in the liquid, their clotting effect would be enhanced, and clotting would begin simultaneously throughout the entire blood volume, distributing the clots evenly throughout the top and bottom of the blood volume alike. The idea is that if the kaolin particles are small enough, they can remain suspended in the blood for a sufficient amount of time before beginning to sink, initiating and accelerating homogeneous clotting, resulting in a homogeneous clot.

[0079] success criteria The kaolin particles should be suspended in the liquid for 15 minutes without sinking.

[0080] method Two particle sizes were tested. Less than 25 microns Over 25 microns

[0081] For each particle size group, 50 mg of powder was placed in a clear glass container containing 160 ml of purified water. Both containers were mixed with a steering spoon for 5 seconds. At 0 and 15 minutes, the containers were visually evaluated for the following parameters: 1. Water color and transparency (from 10: white / opaque to 1: clear / completely transparent) 2. Is there any kaolin powder deposited at the bottom of the container? (10: no deposition to 1: complete deposition)

[0082] result The following table describes the results of this study (also seen in Figures 1A-1B): Table 3 JPEG2025504710000005.jpg79157

[0083] conclusion Kaolin particles smaller than 25 microns will float in water, i.e., blood, for 15 minutes without sinking, whereas kaolin particles larger than 25 microns will sink within 15 minutes.

Claims

1. mixing a quantity of whole blood with calcium gluconate powder and kaolin powder; and 1. A method for preparing a clot for treatment of a subject, comprising clotting the whole blood to form the clot, the method comprising: (i) the amount of calcium gluconate powder to be mixed with the whole blood is approximately 3.3 mg to 7.3 mg per ml of whole blood; (ii) the particle size distribution D50 of the calcium gluconate powder is about 50μ to 200μ; (iii) the amount of kaolin powder is about 0.6 mg to 3.1 mg per ml of whole blood; and (iv) the kaolin powder has a particle size distribution D50 of less than about 25 μm; method.

2. The method described in claim 1, wherein the particle size distribution of the calcium gluconate powder satisfies one of the criteria selected from (i) D70 of approximately 50μ to 200μ, (ii) D90 of approximately 50μ to 200μ, and (iii) D50 of approximately 125μ.

3. 3. The method of claim 1 or 2, wherein the particle size distribution of the kaolin powder meets one of the criteria selected from: (i) D99 less than about 50μ; (ii) D80 less than about 25μ; and (iii) D45 less than about 3μ.

4. The method according to any one of claims 1 to 3, wherein the mixing is carried out in a solidification mold.

5. 5. The method of claim 1, wherein the mixing comprises at least one of the steps of: (i) mixing about 1.75 mg of kaolin powder per 1 ml of whole blood; and (ii) mixing about 5.3 mg of calcium gluconate powder per 1 ml of whole blood.

6. 1. A clotting mold comprising a clotting space defined by a base surrounded by an upwardly facing wall and configured to receive a selected volume of whole blood, the clotting space contains calcium gluconate powder and kaolin powder; The clotting type is (i) the amount of calcium gluconate is about 3.3 mg to 7.3 mg per ml of the selected amount of whole blood; (ii) the particle size distribution D50 of the calcium gluconate powder is about 50μ to 200μ; (iii) the amount of kaolin powder is about 0.6 mg to 3.1 mg per ml of the selected amount of whole blood; and (iv) the kaolin powder has a particle size distribution D50 of less than about 10 μm; Blood clotting type.

7. The blood coagulation type described in claim 6, wherein the particle size distribution of the calcium gluconate powder satisfies one of the criteria selected from (i) D70 of approximately 50μ to 200μ, (ii) D90 of approximately 50μ to 200μ, and (iii) D50 of approximately 125μ.

8. A blood clot type as described in claim 6 or 7, wherein the particle size distribution of the kaolin powder satisfies one of the criteria selected from (i) D99 being less than about 50μ, (ii) D80 being less than about 25μ, and (iii) D45 being less than about 3μ.

9. 9. The blood clot formulation of claim 6, comprising at least one of: (i) about 1.75 mg of kaolin powder per ml of the selected amount of whole blood; and (ii) about 5.3 mg of calcium gluconate powder per ml of the selected amount of whole blood.

10. The clot form according to any one of claims 6 to 9, wherein the volume of the clot space is sufficient to accommodate about 13 ml to 19 ml of whole blood.

11. A kit for preparing a clot from a selected amount of whole blood, comprising a clot type described in any one of claims 6 to 10.

12. 12. The kit of claim 11, comprising a device for collecting and storing whole blood.

13. 13. The kit according to claim 11 or 12, comprising 0.04 ml to 0.095 ml of anticoagulant-containing solution per 1 ml of the selected amount of whole blood.

14. The kit according to any one of claims 11 to 13, wherein the selected volume is between 13 ml and 19 ml.

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