TW202216069A - Kit for isolation of platelet-rich plasma and the method using the same - 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 [mu]m.

Description

High concentration platelet plasma separation kit and method of using the same

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

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

High platelet concentration (PRP) is plasma that contains platelets at higher than baseline levels. It can be activated by the addition of thrombin or calcium chloride (CaCl ₂ ), which will release growth factors, such as fibroblast growth factor-2 (FGF- 2), transforming growth factor-beta 1 (TGF-ß1), bone morphogenic growth factor-2 (bone morphogenic growth factor-2, BMP2), insulin-like growth factor 1 (insulin-like growth factors 1) , IGF1), platelet-derived growth factor (PDGF), and other cytokines. These are valuable in stimulating, signaling, and facilitating the involvement of cells in tissue repair processes. To date, there are various commercially available PRP kits on the market, each kit showing different advantages in the final product, for example, the number of harvested platelets, the percentage of white blood cells or red blood cells, the final content of PRP, the price, and the time of operation . Many commercially available PRP preparation kits on the market follow the same method as the preparation of fibrin glue; it may result in low platelet content, limited platelet count, excess white/red blood cells, and insufficient fibrin ultrastructure. Currently, there are several new PRP devices or kits on the market, which are generally easier to use and more user-friendly than previous kits, although frontline clinicians are still dissatisfied with most PRP kits, either Dissatisfaction with final PRP content, platelet count, growth factor concentration, leukocyte/erythrocyte count, or fibrin ultrastructure.

Commercially available PRP kits can be divided into two categories: plasma-based kits and buffy coat-based kits; which can be briefly described below. Platelets were collected from whole blood by centrifugation according to conventional methods, and the whole blood was divided into three layers: (1) the plasma layer at the top accounted for about 55% of the whole blood and was mainly composed of the least dense components, (2) the buffy coat The layer stays in the middle layer, 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 Plasma-based kits harvest only the top plasma layer with fewer platelets; conversely, the buffy coat kit harvests both the plasma layer and the buffy coat, with a higher concentration of platelets, so-called high-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 the buffy coat set is more popular in tissue repair or regeneration. The action of currently commercially available buffy coat sets is generally based on the density of the blood components. However, the densities of the elements in blood are very similar, and it would be a big challenge to separate the elements in blood only by traditional centrifugation. Therefore, the aforementioned commercially available buffy coat sets are often contaminated with white blood cells, which can cause a local inflammatory response to worsen the healing process, or by red blood cell filtration, which can lead to local cell necrosis. In addition, the number of granular leukocytes in PRP interferes with the formation of fibrin superstructure, such as fibrin diameter, pore size, and porosity, which may be related to cell migration during healing.

In view of this, there is a need for an improved method that can efficiently separate platelets from plasma, specifically from leukocytes and erythrocytes, and obtain high-concentration platelet plasma.

One aspect of the present invention provides a high-concentration platelet-plasma separation kit, comprising: 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 disposed 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 of the present invention is a high-concentration platelet-plasma separation method, comprising: providing a kit of the present invention; placing a blood sample in the first separation tube through the first opening; 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.

Other objects, advantages, and features of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings.

When read in conjunction with the accompanying drawings, the following specific embodiments are made to clearly demonstrate the above and other technical contents, features, and effects of the present invention. Through the description of the specific embodiments, people will further understand the technical means and effects of the present invention adopted to achieve the above-mentioned objects. Furthermore, since those skilled in the art can easily understand and implement the present disclosure, all equivalent changes or modifications without departing from the concept of the present invention will be covered by the scope of the appended claims.

In addition, the ordinal numbers such as "first", "second", etc. described in the description and the scope of the patent application are only intended to describe the claimed elements, and do not imply or indicate that the claimed elements have any progressive ordinal numbers, nor do they imply a The sequence between a claimed element and another claimed element or between steps of a method of manufacture. These ordinal numbers are only used to distinguish one claimed element with a particular name from another claimed element with the same name.

In addition, terms described in the specification and claims, 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 set 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 larger 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 decreases 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 portion 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 disposed on the third opening. Therefore, in the present invention, the first, second, and third cover bodies 110 , 120 , 240 cover the first, second, and third openings 101 , 102 , 201 , respectively.

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

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

In a specific embodiment, the first part 100 and the second part 200 are detachable. However, in another embodiment, the first portion 100 and the second portion 200 are integral.

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

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

Table 1 group abbreviation for isolated product describe Whole blood WB Whole blood collected directly from a donor normal method CP-PRP Whole blood was collected from one donor and centrifuged once at 1600 g to separate the whole blood into three layers. Collect the top two layers and discard the bottom layer. Tubes with 5 µm filter inserts H5M The high-concentration platelet plasma is separated according to the method disclosed in the present invention, wherein the filter membrane has a cut-off pore size of 5 μm. The second separation tube was placed in a centrifuge; leukocytes were then further filtered from the product by centrifugal force of 2300 g. Tubes with 3 µm filters inserted H3M According to the method disclosed in the present invention, the high-concentration platelet plasma is separated, wherein the filter membrane has a cut-off pore size of 3 μm. The second separation tube was placed in a centrifuge; leukocytes were then further filtered from the product by centrifugal force of 2300 g. Tubes with 2 µm filters inserted H2M The high-concentration platelet plasma is separated according to the method disclosed in the present invention, wherein the filter membrane has a cut-off pore size of 2 μm. The second separation tube was placed in a centrifuge; leukocytes were then further filtered from the product by centrifugal force of 2300 g. Commercial kit VB Commercially available PRP kits

PRP is isolated by a two-step method. First, a blood sample is placed in a first separation tube through the first opening. A first centrifugation was performed to separate the bottom layer, the buffy coat, and the top layer in the first separation tube. The bottom layer was discarded from the second opening, and then the buffy coat and the upper layer were transferred to the second separation tube through the third opening, ie, placed in the first portion. A second centrifugation was performed to bring down the plasma in the second separation tube. As a result, plasma passes through the filter and the PRP is collected in the second fraction, while leukocytes are retained in the filter.

Example 2 Complete blood count

Blood cell and platelet concentrations were counted to assess the efficiency of separation. The platelets, white blood cells, and red blood cells of the isolated groups disclosed in Table 1 were counted by an automated veterinary blood analyzer (IDEXX Procyte Dx), respectively, for complete blood count (CBC). The results are shown in Figure 2 and Table 2.

Table 3 summarizes the formulas for enumeration and evaluation of all blood cell components.

Experimental data in this Summary are expressed as mean ± S.D. using GraphPad Prism software 6 unless otherwise stated. Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test, and significance levels were expressed as *p < 0.05, **p < 0.01, and ***p < 0.001.

As shown in FIG. 2 , the platelet concentrations in the CP-PRP group and the WB (whole blood) group were 239×10 ³ /μl and 119×10 ³ /μl, respectively; the former was about twice the concentration of the latter. The platelet counts in the ^H5M group, the H3M group, the H2M group, and the VB group were 8.5×10 ³ /μl, 17.0×10 3 /μl, 22.0×10 ³ /μl, and 13.0×10 ³ /μl, respectively. Among them, the H2M group showed the highest platelet count. Although the platelet concentration of CP-PRP group and WB group was much higher than that of H2M group, from Table 2, the production of white blood cells (WBCs) in CP-PRP group was higher than that in H2M group, H3M group, and H5M group. was much higher in the CP-PRP group, so the PRP collected in the CP-PRP group may have contributed to increased local inflammatory responses. The WB group contains too many WBCs and 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 yield (VY %) Platelet production (PY %) Red blood cell production (RY %) White blood cell production (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 Commercial kit 10 3.833±0.29 38.33±1.44 4.19±0.16 0 5.3

table 3

Table 2 shows a summary of blood cell counts and isolated PRP concentrations. Compared to the commercially available kits of the VB group, the groups according to the invention employing the kits of the present invention, including the H5M group, the H3M group, and the H2M group, collected relatively higher final volumes and PRP from whole blood volume yield.

PRP of the group according to the present invention was successfully and efficiently isolated because the filter only permeates platelets and retains other cells.

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

Therefore, PRP isolated by the kits according to the present invention (H2M, H3M, and H5M) resulted in better volume and platelet concentration, indicating that the kits and methods according to the present invention were effective and successful in isolating from whole blood samples. PRPs, and very little or no red and white blood cells are present in the final product.

Example 3 Growth factor quantification

Growth of PRPs including fibroblast growth factor 2 (FGF-2) and tumor growth factor β-1 (TGF-β1) were detected using an ELISA kit (Wuhan USCN Business Co., Ltd., USA) according to the user's manual. Factor quantification.

Growth factor quantification of the prepared PRPs is shown in Figure 3. The concentrations of growth factors, FGF-2 and TGFβ-1 in the PRPs prepared according to the present invention in the H2M group, the H3M group and the H5M group, and the CP group were all much higher than the average. The concentrations of growth factors in PRPs prepared with other commercially available kits fluctuated widely.

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

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

This result shows that the concentration of growth factors in the group using the kit according to the invention is relatively stable and well above average.

While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only and may be implemented in combination. Numerous variations, changes and substitutions will now occur to those skilled in the art without departing from this invention. It should be understood that various alternatives to the specific embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following patent claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

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

For the purpose of illustrating the invention, embodiments that are presently preferred are shown in the drawings. It should be understood, however, that the invention is not limited to the preferred embodiments shown.

In the schema:

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

Figure 2 shows the platelet count of PRP isolated by the kit of the present invention, the commercial PRP kit, whole blood (WB), and conventional PRP (conventional protocol PRP, CP-PRP).

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

101: The first opening

200: Second separation tube

201: The third opening

210: Part One

220: Part II

230: filter membrane

240: The third cover

Claims (6)

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, comprising a first part, a second part, and a filter membrane; The first part has a third opening, the filter membrane is disposed 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 between in the range of 1.5 to 4 μm. The kit of claim 1, wherein the ratio of the first part and the second part is between 45:55 and 35:65. The kit of claim 1, wherein the first part and the second part are detachable. The kit of 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. A method for separating high-concentration platelet plasma, comprising: provide a kit as described in claim 1; placing a blood sample in the first separation tube through the first opening; 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 The second separation tube is centrifuged to collect the high concentration platelet plasma in the second portion. The method of claim 5, wherein the centrifugation is performed at 2,200 to 2,400 g.

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