TWM618792U - Kit for isolation of platelet-rich plasma - Google Patents

Abstract

Provided herein is a kit for isolation of platelet-rich plasma, comprising a first isolation tube and a second isolation tube. The first isolation tube comprises a first opening and a second opening, and the second isolation tube comprises a first portion, a second portion, and a filter; wherein the first portion has a third opening, the filter is disposed between the first portion and the second portion, the second opening has a diameter in a range of 1 to 5 mm and the filter has a pore size in a range of 1.5 to 4 μm.

Description

High concentration platelet plasma separation kit

The present disclosure relates to a high-concentration platelet plasma separation kit and a method of using the same.

The buffy coat containing high concentration of platelets (Platelet-rich plasma, PRP) and growth factors has been introduced, which has a better effect on tissue repair, especially in bone, cartilage, skin, alveolar tissue, and even as a medicine/ Cell carrier to control release.

High-density platelets (PRP) are plasma containing platelets higher than the baseline content. It can be activated by adding thrombin or calcium chloride (CaCl ₂ ), where thrombin or calcium chloride will release growth factors from alpha particles, such as fibroblast growth factor-2 (FGF- 2), transforming growth factor-beta 1 (TGF-ß1), bone morphogenic growth factor-2 (BMP2), insulin-like growth factor 1 (insulin-like growth factor 1) , IGF1), platelet-derived growth factor (PDGF), and other cytokines. These are valuable in stimulating, transmitting information, and promoting cells to participate in the tissue repair process. So far, there are various commercially available PRP kits on the market, each of which shows different advantages in the final product, such as the number of platelets harvested, the percentage of white blood cells or red blood cells, the final content of PRP, price, and operating time . Many commercially available PRP preparation kits on the market follow the same method as the preparation of fibrin glue; it may cause low platelet content, limited number of platelets, excessive white blood cells/red blood cells, and insufficient fibrin ultrastructure. Currently, there are several new PRP equipment or sets on the market. They are generally easier to use and user-friendly than the previous sets. Although the first-line clinical staff are still not satisfied with most PRP sets, whether they are Dissatisfied with the final content of PRP, the number of platelets, the concentration of growth factors, the number of white blood cells/red blood cells, or the ultrastructure of fibrin.

Commercially available PRP kits can be divided into two categories: plasma-based kits and buffy coat-based kits; which can be briefly described as follows. The platelets are collected from the whole blood by centrifugation according to the conventional method, and the whole blood is divided into three layers: (1) The plasma layer on the top accounts for about 55% of the whole blood, mainly composed of the least dense components, and (2) Erythrocyte sedimentation rate The layer stays in the middle layer, which is less than 1% of the whole blood, and has a high concentration of white blood cells and platelets. (3) The red blood cells at the bottom account for about 45% of the whole blood. ¹⁶ The plasma-based kit only harvests the top plasma layer, and there are fewer platelets; on the contrary, the buffy coat set harvests the plasma layer and the buffy coat, which has a higher platelet concentration, the so-called high-concentration platelet plasma, Referred to as PRP. Platelets can release more growth factors to induce local cells or mesenchymal stem cells to participate in the repair process during tissue healing; therefore, buffy coat sets are more popular in tissue repair or regeneration. The effect of currently commercially available buffy coat sets is usually based on the density of blood components. However, the density of each element in the blood is very close, and it would be a big challenge to separate the elements in the blood only by traditional centrifugation. Therefore, the aforementioned buffy coat sets on the market are usually contaminated with white blood cells, which may cause local inflammation to worsen the healing process, or be contaminated by red blood cell filtration, which may cause local cell necrosis. In addition, the number of granular white blood cells in PRP can interfere with the formation of fibrin superstructures, such as fibrin diameter, pore size, and porosity, which may be related to cell migration during the healing process.

In view of this, there is a need for an improved method that can effectively separate platelets from plasma, specifically separating platelets from white blood cells and red blood cells, and obtain high-concentration platelet plasma.

One aspect of the present disclosure is a high-concentration platelet plasma separation kit, including: a first separation tube and a second separation tube. The first separation tube includes a first opening and a second opening, the second separation tube includes a first part, a second part, and a filter membrane; wherein the first part has a third opening, and the filter membrane is arranged Between the first part and the second part, the diameter of the second opening is in the range of 1 to 5 mm, and the pore size of the filter membrane is in the range of 1.5 to 4 μm.

Another aspect provided by the present disclosure is a method for separating high-concentration platelet plasma, comprising: providing the set of the present disclosure; placing a blood sample in the first separation tube through the first opening; and centrifuging the first separation tube To separate a bottom layer, a buffy coat, and an upper layer; discard the bottom layer from the second opening; transfer the buffy coat and the upper layer to the second separation tube through the third opening; and centrifuge the first Two separate tubes to collect the high-concentration platelet plasma in the second part.

When combined with the drawings, other purposes, advantages, and features of the present disclosure will become more apparent according to the following detailed description.

When read in conjunction with the drawings, the following specific embodiments are made to clearly demonstrate the above and other technical content, features, and effects of the disclosure. Through the description of specific specific embodiments, people will further understand the technical means and effects of the present disclosure used to achieve the above-mentioned objectives. In addition, since those skilled in the art can easily understand and implement the content disclosed in this disclosure, all equivalent changes or modifications without departing from the concept of this disclosure will be covered by the scope of the attached patent application.

In addition, the ordinal numbers described in the specification and the scope of the patent application, such as "first", "second", etc., are only used to describe the claimed element, and do not imply or indicate that the claimed element has any sequential ordinal number, nor does it imply a The sequence between a claimed element and another claimed element or between steps of a manufacturing method. These ordinal numbers are only used to distinguish one claimed element with a specific name from another claimed element with the same name.

In addition, the terms described in the specification and the scope of the patent application, such as "on" not only mean direct contact with another element, but also mean indirect contact with another element.

High Concentration Platelet Plasma (PRP) Separation Kit

Please refer to Figure 1 and Figure 2. The PRP separation kit includes a first separation tube 100 and a second separation tube 200. The first separation tube 100 has a first opening 101 and a second opening 102. In a preferred embodiment, the diameter of the first opening 101 is greater than the diameter of the second opening 102. In a more preferred embodiment, the diameter of the second opening 102 ranges from 1 to 5 mm. A first cover 110 is disposed on the first opening 101, and a second cover 120 is disposed on the second opening 102. As shown in FIG. 1, the diameter of the first separation tube 100 is reduced near the second opening 102 and thus forms a pointed shape.

The second separation tube 200 includes a first part 210, a second part 220, and a filter membrane 230. The first part 210 has a third opening 201. The first part 210 and the second part 220 are connected to each other, and the filter membrane 230 is disposed between the first part 210 and the second part 220. The pore size of the filter membrane 230 is in the range of 1.5 to 4 μm. A third cover is arranged on the third opening. Therefore, in the present disclosure, the first, second, and third covers 110, 120, and 240 will cover the first, second, and third openings 101, 102, and 201, respectively.

In a specific embodiment, the ratio of the first part 210 to the second part 220 is between 45:55 and 35:65. In a preferred embodiment, the ratio of the first part 210 to the second part 220 is about 40:60.

In a specific embodiment, the length of the first separation tube 100 and the second separation tube 200 is between 10 and 20 cm.

In a specific embodiment, the first part 210 and the second part 220 are detachable. However, in another specific embodiment, the first part 210 and the second part 220 are integrated.

Example 1 Preparation of blood samples and separation of high-concentration platelet plasma (PRP)

The total volume of whole blood collected from pigs was 500 ml by supplementing ACD-A anticoagulant (GBiosciences, USA) through internal jugular vein puncture. As shown in Table 1, the blood samples were divided into eight groups to separate PRP through different methods.

Table 1 Group Abbreviation for separation product describe Whole blood WB Whole blood collected directly from a donor normal method CP-PRP Collect whole blood from a donor and perform a centrifugation with a centrifugal force of 1600 g to divide the whole blood into three layers. Collect the top two layers and discard the bottom layer. Test tube with 5 µm filter membrane H5M According to the method disclosed in the present disclosure, high-concentration platelet plasma is separated, wherein the filter membrane has a cut-off pore size of 5 μm. The second separation tube is placed in a centrifuge; then the white blood cells are further filtered out of the product through the centrifugal force of 2300 g. Test tube with 3 µm filter membrane H3M According to the method disclosed in the present disclosure, high-concentration platelet plasma is separated, wherein the filter membrane has a cut-off pore size of 3 μm. The second separation tube is placed in a centrifuge; then the white blood cells are further filtered out of the product through the centrifugal force of 2300 g. Test tube with 2 µm filter membrane H2M According to the method disclosed in the present disclosure, high-concentration platelet plasma is separated, wherein the filter membrane has a cut-off pore size of 2 μm. The second separation tube is placed in a centrifuge; then the white blood cells are further filtered out of the product through the centrifugal force of 2300 g. Commercially available sets VB Commercially available PRP sets

A two-step method is used to separate PRP. First, a blood sample is placed in a first separation tube through the first opening. Perform the first centrifugation to separate the bottom layer, the buffy coat layer, and the upper layer in the first separation tube. The bottom layer is discarded from the second opening, and then the buffy coat and the upper layer are transferred to the second separation tube through the third opening, that is, placed in the first part. Perform a second centrifugation to drop the plasma in the second separation tube. As a result, the plasma passes through the filter membrane and the PRP is collected in the second part, while the white blood cells are retained in the filter membrane.

Example 2 Complete blood count

Count the concentration of blood cells and platelets to evaluate the efficiency of separation. In the separation groups disclosed in Table 1, platelets, white blood cells, and red blood cells were counted through an automatic veterinary blood analyzer (IDEXX Procyte Dx) to perform a complete blood count (CBC). The results are shown in Figure 2 and Table 2.

Table 3 summarizes the formula for counting and evaluating all blood cell components.

Unless otherwise specified, GraphPad Prism software 6 is used to express the experimental data in the content of this disclosure as an average ± S.D. The data was analyzed by one-way ANOVA, and then the Tukey multiple comparison test was performed. The degree of significance is expressed as *p <0.05, ** p <0.01, and *** p <0.001.

As shown in Figure 2, the platelet concentrations of the CP-PRP group and the WB (whole blood) group were 239×10 ³ cells/μl and 119×10 ³ cells/μl, respectively; the former was about twice the concentration of the latter. H5M group, platelet counts H3M group, H2M group, VB group and were /μl,17.0x10 ³ 8.5x10 ³ th th th /μl,22.0x10 ³ / μl, and th 13.0x10 ³ / μl. Among them, the H2M group showed the highest number of platelets. Although the platelet concentration of the CP-PRP group and the WB group was much higher than that of the H2M group, from Table 2, the white blood cell (WBCs) production in the CP-PRP group was higher than that of the H2M group, H3M group, and H5M group. It is much higher in the group, so the PRP collected in the CP-PRP group may cause an increase in local inflammation. The WB group contains too much WBC and red blood cells (red blood cells, RBCs), which may not only cause local inflammation, but also slow down the healing process.

Table 2 PRP set Initial volume (ml) Final volume (ml) Volume production (VY %) Platelet production (PY %) Red blood cell production (RY %) White blood cell yield (WY %) CP-PRP 30 15.65±1.32 50.24±4.40 100.90±8.83 0.18±0.03 27.83±15.94 H5M 4 1.93±0.09 48.13±2.29 3.44±0.16 0.05±0.02 0 H3M 4 1.82±0.09 45.39±2.37 6.48±0.34 0.01±0.00 0 H2M 4 1.82±0.09 45.39±2.37 8.39±0.44 0 0 Commercially available sets 10 3.833±0.29 38.33±1.44 4.19±0.16 0 5.3

table 3

Table 2 shows a summary of the blood cell count and the isolated PRP concentration. Compared with the commercially available kits of the VB group, the groups according to the disclosure using the kits of the disclosure include the H5M group, the H3M group, and the H2M group. A relatively higher final volume and PRP are collected from whole blood. The volume yield.

The PRP of the group according to the present disclosure was successfully and effectively separated because the filter membrane only allows platelets to permeate and retains other cells.

The H2M group, H3M group, and H5M group according to the present disclosure can effectively separate RBC and WBC from PRP, especially the H2M group. In addition, the RBC in the H5M group and the H3M group were reduced to 0.05% and 0.01%, respectively, which was significantly lower than the CP-PRP (conventional method) group.

Therefore, PRP separated by the kit (H2M, H3M, and H5M) according to the present disclosure produces better volume and platelet concentration, which means that the kit and method according to the present disclosure are effectively and successfully separated from whole blood samples PRPs, and there are only very low or even no red blood cells and white blood cells in the final product.

Example 3 Growth factor quantification

Use the ELISA kit (Wuhan USCN Business Co., Ltd., USA) to grow PRPs including fibroblast growth factor 2 (FGF-2) and tumor growth factor β-1 (TGF-β1) according to the user manual Factor quantification.

The growth factor quantification of the prepared PRPs is shown in Figure 3. According to the present disclosure, the concentrations of growth factors, FGF-2 and TGFβ-1 in the PRPs prepared in the H2M group, H3M group, H5M group, and CP group are much higher than the average. The concentration of growth factors in PRPs prepared with other commercially available kits fluctuates greatly.

As for the concentration of FGF-2 (Figure 3(A)), the concentrations of the H5M group, H3M group, H2M group, and CP group were all higher than 750 pg/ml. The concentration in the VB group was less than 500 pg/ml.

As for the concentration of TGFβ-1 (Figure 3(B)), the concentrations of the H5M group, H3M group, H2M group, and CP group were all higher than 1750 pg/ml. The concentration in the VB group was lower than 1500 pg/ml.

This result indicates that the concentration of growth factors in the group using the set according to the present disclosure is relatively stable and much higher than the average.

Although the preferred specific embodiments of the present disclosure have been shown and described in the present disclosure, it is obvious to those skilled in the art that these specific embodiments are only provided by way of example and can be implemented in combination. Without departing from this disclosure, those skilled in the art will now think of many changes, changes and substitutions. It should be understood that various alternatives to the specific embodiments of the present disclosure described in this disclosure can be used to implement the present disclosure. The intention is to limit the scope of the disclosure with the scope of the following patent applications, and thus cover the methods and structures within the scope of these patent applications and their equivalents.

100: The first separation tube 101: first opening 102: second opening 110: The first cover 120: second cover 200: The second separation tube 201: third opening 210: Part One 220: Part Two 230: filter membrane 240: third cover

To illustrate the present disclosure, the presently preferred embodiments are shown in the drawings. However, it should be understood that the present disclosure is not limited to the preferred embodiments shown.

In the schema:

Figure 1 shows a schematic diagram of the kit according to the present disclosure; (A) the first separation tube; (B) the second separation tube.

Figure 2 shows the number of platelets of PRP separated by the kit of the present disclosure, the commercially available PRP kit, whole blood (WB), and the conventional method PRP (conventional protocol PRP, CP-PRP).

Figure 3 shows the concentration of growth factors released from activated PRP in all groups listed in Table 1: (A) FGF-2, (B) TGF-β1; the same labels (a, b, c, Or d) indicates that there is no statistical difference, and the degree of significance is expressed as * (p <0.05), ** (p <0.01), and *** (p <0.001).

101: first opening

200: The second separation tube

201: third opening

210: Part One

220: Part Two

230: filter membrane

240: third cover

Claims (4)

A high-concentration platelet plasma separation kit, comprising: a first separation tube including a first opening and a second opening, and a second separation tube including a first part, a second part, and a filter membrane; The first part has a third opening, the filter membrane is arranged between the first part and the second part, the diameter of the second opening is in the range of 1 to 5 mm, and the pore size of the filter membrane is 1.5 The second opening corresponds to the third opening, and the platelet plasma separated from the first separation tube flows out through the second opening, and then is injected into the second separation tube from the third opening. The set according to claim 1, wherein the ratio of the first part and the second part is between 45:55 and 35:65. The set according to claim 1, wherein the first part and the second part are detachable. The set according to claim 1, further comprising a first cover, a second cover, and a third cover to cover the first opening, the second opening, and the third opening, respectively.

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