技术领域technical field
本发明属临床医学技术领域，具体涉及一种提取富含血小板的血浆的方法，本发明方法在特定条件下获得的富血小板血浆具有血小板浓度高、血小板回收率高等至少一个方面的优点。The invention belongs to the technical field of clinical medicine, and specifically relates to a method for extracting platelet-rich plasma. The platelet-rich plasma obtained under specific conditions by the method of the invention has at least one advantage of high platelet concentration and high platelet recovery rate.
背景技术Background technique
富含血小板的血浆，在本发明中可称为富血小板血浆(Platelet RichPlasma，PRP)，其是是通过离心血液而得到的含高浓度的血小板血浆。人体的血小板除了可以在止血的时候提供凝聚作用外，血小板中还含有很多与创伤愈合和骨头再生有关的多种生长因子。如血小板衍生生长因子(Platelet Derived Growth Factor，PDGF)，转化生长因子(Transforming Growth Factor，TGF)，胰岛素样生长因子(Insulin-likeGrowth Factor，IGF)，表皮生长因子(Epidermal Growth Factor，EGF)以及血管内皮生长因子(Vascular Endothelial Growth Factor，VEGF)等等，血小板来源的生长因子，对细胞的分化和增殖起着促进作用，这些生长因子相互之间具有协同响应，与其他促进细胞活性的因子相互作用和影响，共同维持着组织环境的平衡，对伤口愈合、修复和再生有着重要的作用。Platelet-rich plasma may be referred to as platelet-rich plasma (Platelet Rich Plasma, PRP) in the present invention, which is high-concentration platelet-containing plasma obtained by centrifuging blood. In addition to providing coagulation during hemostasis, platelets in the human body also contain many growth factors related to wound healing and bone regeneration. Such as platelet-derived growth factor (Platelet Derived Growth Factor, PDGF), transforming growth factor (Transforming Growth Factor, TGF), insulin-like growth factor (Insulin-like Growth Factor, IGF), epidermal growth factor (Epidermal Growth Factor, EGF) and vascular Endothelial growth factor (Vascular Endothelial Growth Factor, VEGF), etc., growth factors derived from platelets, promote cell differentiation and proliferation. These growth factors have a synergistic response to each other and interact with other factors that promote cell activity. Together, they maintain the balance of the tissue environment and play an important role in wound healing, repair and regeneration.
另外，由于PRP可以从患者自身血液中提取，经提取富集处理后，可用于相关疾病的治疗，因而来源丰富，取材方便，制备方法简单而且可以让身体吸收。自身PRP使用能避免病毒传播和免疫排斥的问题出现。因此PRP被广泛应用到不同的医学领域中。如骨科、口腔科、颌面外科、运动医学以及美容医学。In addition, because PRP can be extracted from the patient's own blood, after extraction and enrichment, it can be used for the treatment of related diseases, so the source is rich, the material is convenient, the preparation method is simple, and it can be absorbed by the body. The use of self-PRP can avoid the problems of virus transmission and immune rejection. Therefore, PRP is widely used in different medical fields. Such as orthopedics, dentistry, maxillofacial surgery, sports medicine and aesthetic medicine.
PRP在目前为止还没有一个统一的制备方法，PRP的制备原理主要是利用血液中各种成分沉降速度不同，通过离心将血液分层。从而获取含血小板的血浆，并利用离心原理进一步的浓缩以获得高浓度的血小板血浆。人体全血中血小板的浓度一般为1~3×105/ml。研究认为浓缩后的PRP血小板浓度应为全血血小板浓度的3-4倍。研究也发现PRP中血小板浓度的提高能有效提高干细胞的增殖和分化能力以及能够显著增加成纤维细胞的增殖和I型胶原蛋白的表达。So far, there is no unified preparation method for PRP. The main principle of PRP preparation is to use the different sedimentation speeds of various components in the blood to separate the blood into layers by centrifugation. In this way, platelet-containing plasma is obtained, and further concentrated by centrifugation to obtain high-concentration platelet plasma. The concentration of platelets in human whole blood is generally 1~3×105/ml. Studies suggest that the concentrated PRP platelet concentration should be 3-4 times the whole blood platelet concentration. Studies have also found that increasing the concentration of platelets in PRP can effectively improve the proliferation and differentiation capabilities of stem cells and can significantly increase the proliferation of fibroblasts and the expression of type I collagen.
因此有效的分离和提高PRP中血小板的浓度成为了整个PRP治疗方案的关键。Therefore, effectively separating and increasing the concentration of platelets in PRP has become the key to the entire PRP treatment plan.
林庶茹等(CN101402940A，中国专利申请号：200810228726.5，发明名称：一种小鼠血小板的提取方法)公开了一种小鼠血小板的提取方法，小鼠眶静脉取血，置于抗凝离心管中，静置30MIN后离心，800RPM离心10MIN，上清液即为富含血小板的血浆。吸出上清液置于干净试管中，3500RPM离心10MIN，弃去上清液，管底沉淀物为血小板。向试管中滴加50～100ΜL质量浓度为1%草酸铵溶液，以玻璃棒轻轻搅拌，再加入质量浓度为1%草酸铵溶液2～3ML，静置5MIN，使红细胞溶解。3500RPM离心10MIN，弃去上清，加入血小板洗涤液并反复吹打，使成为血小板混悬液，3300RPM离心10MIN，弃去上清，加入少量血小板洗涤液并反复吹打使之成为混悬液。该血小板将作为抗原免疫豚鼠后，取豚鼠血清给小鼠注射，制备过敏性紫癜模型。此方法操作简单，可以得到高浓度的血小板，并很好地解决血小板纯度不佳的问题。这种针对小鼠血液进行血小板富集的方法虽然据信可以获得浓度达1010的血小板，然而该方法未公开其血小板的收率，并且方法在操作上比较繁杂，难以适用于人血的处理。(CN101402940A, Chinese patent application number: 200810228726.5, title of invention: a method for extracting mouse platelets) discloses a method for extracting mouse platelets. Blood is taken from the orbital vein of mice and placed in an anticoagulant centrifuge tube. Centrifuge after standing for 30min, then centrifuge at 800RPM for 10min, the supernatant is platelet-rich plasma. Aspirate the supernatant and place it in a clean test tube, centrifuge at 3500RPM for 10min, discard the supernatant, the sediment at the bottom of the tube is platelets. Add 50-100 ML of 1% ammonium oxalate solution dropwise to the test tube, stir gently with a glass rod, then add 2-3 mL of 1% ammonium oxalate solution, and let it stand for 5 minutes to dissolve the red blood cells. Centrifuge at 3500RPM for 10min, discard the supernatant, add platelet washing solution and pipette repeatedly to make a platelet suspension, centrifuge at 3300RPM for 10min, discard the supernatant, add a small amount of platelet washing solution and pipette repeatedly to make a suspension. After the platelets are used as an antigen to immunize guinea pigs, the serum of guinea pigs is taken and injected into mice to prepare a model of allergic purpura. This method is simple to operate, can obtain high-concentration platelets, and solves the problem of poor platelet purity. Although this method of platelet enrichment for mouse blood is believed to be able to obtain platelets with a concentration of 1010 , the yield of platelets is not disclosed in this method, and the method is complicated in operation and difficult to apply to the treatment of human blood .
此外，王悦等(CN102078644A，中国专利申请号201110053979.5，发明名称：一种简便高效的自体富血小板血浆提取装置及提取方法)中公开了一种简便高效的自体富血小板血浆提取装置及提取方法,所提取的自体富血小板血浆中含有多种复合生长因子,能够有效地促进组织的修复。由1个注射器、1个静脉输液器连接管、1个静脉留置针塑料卡片和1个静脉输液器针头组成;注射器用以抽取静脉血并充当离心管;静脉输液器针头通过静脉输液器连接管和注射器出口连接,静脉输液器针头用以皮肤血管穿刺、抽取抗凝剂和激活剂并向人体需要部位注射提取后的自体富血小板血浆;静脉输液器连接管可弯曲固定于静脉留置针塑料卡片中。据信这种装置在提升血小板提取的操作性方面是有益的。In addition, Wang Yue et al. (CN102078644A, Chinese Patent Application No. 201110053979.5, Invention Name: A Simple and Efficient Autologous Platelet-Rich Plasma Extraction Device and Extraction Method) disclosed a simple and efficient autologous platelet-rich plasma extraction device and extraction method, The extracted autologous platelet-rich plasma contains a variety of complex growth factors, which can effectively promote tissue repair. Consists of 1 syringe, 1 intravenous infusion set connecting tube, 1 intravenous indwelling needle plastic card and 1 intravenous infusion set needle; the syringe is used to draw venous blood and act as a centrifuge tube; the intravenous infusion set needle passes through the intravenous infusion set connecting tube Connected with the outlet of the syringe, the needle of the venous infusion set is used to puncture blood vessels in the skin, extract anticoagulants and activators, and inject the extracted autologous platelet-rich plasma to the required parts of the human body; the connecting tube of the venous infusion set can be bent and fixed on the plastic card of the venous indwelling needle middle. This device is believed to be beneficial in improving the operability of platelet extraction.
因此为了从血液中富集血小板而得到有临床治疗意义的富血小板血浆，探寻一种操作过程简单、收率高、富集的血浆中血小板浓度高的方法，是本领域技术人员迫切期待的。Therefore, in order to obtain platelet-rich plasma with clinical significance by enriching platelets from blood, it is urgently desired by those skilled in the art to find a method with simple operation, high yield and high platelet concentration in the enriched plasma.
发明内容Contents of the invention
本发明的目的是探索一种简单和高效地分离和浓缩血浆中血小板的方法；特别是本发明为了从血液中富集血小板而得到有临床治疗意义的富血小板血浆，探寻一种操作过程简单、收率高、富集的血浆中血小板浓度高的方法。本发明人发现利用梯度离心法进行血小板的浓缩和分离，在特定操作条件下可以以高收率地获得含浓度高血小板的血浆。The purpose of the present invention is to explore a simple and efficient method for separating and concentrating platelets in blood plasma; especially in order to obtain platelet-rich plasma with clinical significance for enriching platelets from blood, the present invention seeks a method with simple operation process, A method with high yield and high platelet concentration in the enriched plasma. The present inventors found that using gradient centrifugation to concentrate and separate platelets, plasma containing high platelets can be obtained with high yield under specific operating conditions.
为此，本发明第一方面提供了一种从血液中提取富血小板血浆的方法，其包括以下步骤：To this end, the first aspect of the present invention provides a method for extracting platelet-rich plasma from blood, which includes the following steps:
(a)使采集的全血置于含有抗凝剂的容器中，使血液和抗凝剂充分混匀；(a) Place the collected whole blood into a container containing an anticoagulant to fully mix the blood and the anticoagulant;
(b)使混有抗凝剂的血液置于离心管中，进行第一次离心，使血液基本上分成三层；(b) placing the blood mixed with an anticoagulant in a centrifuge tube, and performing the first centrifugation so that the blood is basically divided into three layers;
(c)将最上层和大部分的中间层抽出并转移到新的离心管中，混合均匀，进行第二次离心；(c) The uppermost layer and most of the middle layer are taken out and transferred to a new centrifuge tube, mixed evenly, and centrifuged for the second time;
(d)弃掉离心管上层的血浆，利用剩余的血浆使沉淀的血小板重新悬浮，即得富血小板血浆(Platelet Rich Plasma：PRP)。(d) Discard the plasma in the upper layer of the centrifuge tube, and use the remaining plasma to resuspend the precipitated platelets to obtain platelet rich plasma (Platelet Rich Plasma: PRP).
根据本发明第一方面的方法，其中步骤(a)中所述全血是新鲜的全血。The method according to the first aspect of the present invention, wherein said whole blood in step (a) is fresh whole blood.
根据本发明第一方面的方法，其中步骤(a)中所述全血是受试个体例如患者的全血。由此，本发明获得的富血小板血浆可以方便地重新用于该患者以治疗相关疾病。The method according to the first aspect of the present invention, wherein said whole blood in step (a) is whole blood of a subject such as a patient. Thus, the platelet-rich plasma obtained in the present invention can be conveniently reused for the patient to treat related diseases.
根据本发明第一方面的方法，其中步骤(a)中抗凝剂选自乙二胺四乙酸(Ethylene Diamine Tetraacetic Acid：EDTA)、乙二胺四乙酸二钠、乙二胺四乙酸钙钠、枸橼酸葡萄糖(Acid Citrate Dextrose-Solution A：ACD-A)、或其组合。According to the method of the first aspect of the present invention, wherein in the step (a), the anticoagulant is selected from ethylenediaminetetraacetic acid (Ethylene Diamine Tetraacetic Acid: EDTA), disodium ethylenediaminetetraacetic acid, calcium sodium edetate, Citrate Dextrose-Solution A (ACD-A), or a combination thereof.
根据本发明第一方面的方法，其中步骤(a)中抗凝剂与全血的重量比为1：8~10，例如1：9。The method according to the first aspect of the present invention, wherein the weight ratio of anticoagulant to whole blood in step (a) is 1:8-10, for example 1:9.
根据本发明第一方面的方法，其中步骤(a)中所述容器可以是离心管或是采血袋等。如果是离心管并且该离心管容量适合直接离心，则所述步骤(b)中不需要另置于离心管中，而可以直接进行离心。According to the method of the first aspect of the present invention, wherein the container in step (a) can be a centrifuge tube or a blood collection bag, etc. If it is a centrifuge tube and the capacity of the centrifuge tube is suitable for direct centrifugation, then the step (b) does not need to be placed in a centrifuge tube, but can be centrifuged directly.
根据本发明第一方面的方法，其中步骤(a)中还包括在使血液和抗凝剂后取适量(例如少于500ul，例如约100ul)用于测定其中血液中血小板的数量，以便用于后续的过程比照、监控。According to the method of the first aspect of the present invention, wherein step (a) also includes taking an appropriate amount (such as less than 500ul, such as about 100ul) after making the blood and the anticoagulant for determining the number of platelets in the blood, so as to be used Subsequent process comparison and monitoring.
根据本发明第一方面的方法，其中步骤(b)中经第一次离心后是达到使血液基本上分成三层的程度。在一个实施方案中，其中最上层是含血小板的血浆层，中间一层是血块黄层(Buffy Coat层，其中含有血小板和白细胞)，最底层的是红细胞层。According to the method of the first aspect of the present invention, wherein in step (b), after the first centrifugation, the blood is substantially divided into three layers. In one embodiment, the uppermost layer is a platelet-containing plasma layer, the middle layer is a buffy coat (Buffy Coat layer, which contains platelets and white blood cells), and the bottom layer is a red blood cell layer.
根据本发明第一方面的方法，其中步骤(b)中离心的转速为2000rpm~3000rpm，例如2200rpm~2800rpm，例如2300rpm~2500rpm，例如约2400rpm。According to the method of the first aspect of the present invention, wherein the rotational speed of centrifugation in step (b) is 2000rpm~3000rpm, for example 2200rpm~2800rpm, for example 2300rpm~2500rpm, for example about 2400rpm.
根据本发明第一方面的方法，其中步骤(b)中离心的时间为1-10min，例如2-8min，例如3-5min，例如约4min。The method according to the first aspect of the present invention, wherein the centrifugation time in step (b) is 1-10 min, such as 2-8 min, such as 3-5 min, such as about 4 min.
根据本发明第一方面的方法，其中步骤(b)中离心的转速为2300rpm~2500rpm，时间为3-5min。According to the method of the first aspect of the present invention, wherein the centrifugation speed in step (b) is 2300rpm-2500rpm, and the time is 3-5min.
根据本发明第一方面的方法，其中步骤(b)中离心的转速为2400rpm，时间为4min。According to the method of the first aspect of the present invention, wherein the rotating speed of centrifugation in step (b) is 2400rpm, and the time is 4min.
根据本发明第一方面的方法，其中步骤(c)中，所述大部分的中间层是指尽量将中间层全部吸出但避免抽取红细胞。According to the method of the first aspect of the present invention, wherein in step (c), said most of the middle layer refers to aspirating all the middle layer as much as possible but avoiding extraction of red blood cells.
根据本发明第一方面的方法，其中步骤(c)中离心的转速为1000rpm~2000rpm，例如1200rpm~1800rpm，例如1400rpm~1600rpm，例如约1500rpm。According to the method of the first aspect of the present invention, wherein the rotational speed of centrifugation in step (c) is 1000rpm~2000rpm, for example 1200rpm~1800rpm, for example 1400rpm~1600rpm, for example about 1500rpm.
根据本发明第一方面的方法，其中步骤(c)中离心的时间为10-30min，例如15-25min，例如18-22min，例如约20min。The method according to the first aspect of the present invention, wherein the centrifugation time in step (c) is 10-30 min, such as 15-25 min, such as 18-22 min, such as about 20 min.
根据本发明第一方面的方法，其中步骤(c)中离心的转速为1400rpm~1600rpm，时间为18-22min。According to the method of the first aspect of the present invention, wherein the centrifugation speed in step (c) is 1400rpm~1600rpm, and the time is 18-22min.
根据本发明第一方面的方法，其中步骤(c)中离心的转速为1500rpm，时间为20min。According to the method of the first aspect of the present invention, wherein the rotating speed of centrifugation in step (c) is 1500rpm, and the time is 20min.
根据本发明第一方面的方法，其中步骤(c)中所得上层和大部分中间层经混匀后，其中血小板的浓度是分离前全血中血小板浓度的1-5倍，例如1.5~4倍，例如2~3倍。According to the method of the first aspect of the present invention, wherein after the upper layer and most of the middle layer obtained in step (c) are mixed, the concentration of platelets is 1-5 times, such as 1.5 to 4 times, the concentration of platelets in the whole blood before separation , for example 2~3 times.
根据本发明第一方面的方法，其中步骤(c)中还包括在使抽取的最上层和中间层混合均匀后，取适量(例如少于500ul，例如约100ul)用于测定其中的血小板的数量，以便用于后续的过程比照、监控。According to the method of the first aspect of the present invention, wherein step (c) also includes taking an appropriate amount (such as less than 500ul, such as about 100ul) for determining the number of platelets therein after mixing the extracted uppermost layer and the middle layer evenly , so as to be used for subsequent process comparison and monitoring.
根据本发明第一方面的方法，其中步骤(d)中，所述弃掉离心管上层的血浆是指弃掉离心管上层至少2/4的血浆。在一个实施方案中，在步骤(d)中，所述弃掉离心管上层的血浆是指弃掉离心管上层至少3/4的血浆。在此应当说明，上层血浆中仅含有低浓度的血小板。According to the method of the first aspect of the present invention, wherein in step (d), discarding the plasma in the upper layer of the centrifuge tube refers to discarding at least 2/4 of the plasma in the upper layer of the centrifuge tube. In one embodiment, in step (d), discarding the plasma in the upper layer of the centrifuge tube refers to discarding at least 3/4 of the plasma in the upper layer of the centrifuge tube. It should be noted here that the upper plasma contains only low concentrations of platelets.
根据本发明第一方面的方法，其中步骤(d)中，弃掉离心管上层的血浆后，可以利用下部剩下的(约2/4，或者更优选的约1/4)血浆重新使沉淀下来的血小板悬浮，由此可以获取本发明所述富血小板血浆(PRP)。According to the method of the first aspect of the present invention, wherein in step (d), after discarding the plasma on the upper layer of the centrifuge tube, the remaining (about 2/4, or more preferably about 1/4) plasma in the lower part can be used to re-precipitate The platelets that come down are suspended, so that the platelet-rich plasma (PRP) of the present invention can be obtained.
根据本发明第一方面的方法，其中步骤(d)所得富血小板血浆(PRP)中血小板的浓度是分离前全血中血小板浓度的5-10倍，例如6~8倍，例如6~7倍。According to the method of the first aspect of the present invention, wherein the concentration of platelets in the platelet-rich plasma (PRP) obtained in step (d) is 5-10 times, such as 6-8 times, such as 6-7 times, the concentration of platelets in whole blood before separation .
根据本发明第一方面的方法，其中步骤(d)中还包括在获得富血小板血浆(PRP)后，取适量(例如少于500ul，例如约100ul)用于测定其中的血小板的数量。According to the method of the first aspect of the present invention, step (d) further includes taking an appropriate amount (for example, less than 500ul, such as about 100ul) to determine the number of platelets in the platelet-rich plasma (PRP).
本发明由此获得的富血小板血浆(PRP)具有浓度高的特点，并且血小板回收率高。The platelet-rich plasma (PRP) thus obtained in the present invention has the characteristics of high concentration and high platelet recovery rate.
本发明第二方面提供了一种富血小板血浆，其中包含0.5~3×109/ml浓度的血小板。例如其中包含1~2×109/ml浓度的血小板。The second aspect of the present invention provides platelet-rich plasma, which contains platelets at a concentration of 0.5-3×109 /ml. For example, it contains platelets at a concentration of 1~2×109 /ml.
根据本发明第二方面的富血小板血浆，其基本上是由本发明第一方面任一实施方案所述的方法获得的。The platelet-rich plasma according to the second aspect of the present invention is basically obtained by the method described in any embodiment of the first aspect of the present invention.
下面对本发明作进一步的说明。本发明所引用的文献，以及该文献中所引用的文献，它们的全部内容通过引用并入本文。The present invention will be further described below. The documents cited in this application, as well as the documents cited in this document, are hereby incorporated by reference in their entirety.
在本发明中，本发明任一方面的任一技术方案中，其任一技术特征同样适用于本发明的任一方面的任一实施方案，只要它们不会引起矛盾，并且这种相互适用在必要时可以作适当的修改。In the present invention, in any technical solution of any aspect of the present invention, any technical feature thereof is also applicable to any implementation of any aspect of the present invention, as long as they do not cause conflicts, and this mutual application is Appropriate modifications can be made if necessary.
在本发明一个实施方案中，所述的全血是人的全血。In one embodiment of the invention, said whole blood is human whole blood.
在本发明中，提供了一种采用特定处置条件进行血小板富集的方法。然而本领域技术人员理解，使用本发明的处置条件，其中所用的器具是可以作适当变动的，例如其中的离心装置。例如可以使用王悦等(CN102078644A，中国专利申请号201110053979.5，发明名称：一种简便高效的自体富血小板血浆提取装置及提取方法)中所用的由注射器、静脉输液器连接管、静脉留置针塑料卡片和静脉输液器针头组成的装置，该文献所记载的装置可以用于本发明方法，并使本发明方法在保持操作过程的洁净度中是有益的。In the present invention, a method for platelet enrichment using specific treatment conditions is provided. However, those skilled in the art understand that, using the treatment conditions of the present invention, the instruments used therein can be appropriately changed, such as the centrifuge device therein. Such as Wang Yue etc. (CN102078644A, Chinese patent application number 201110053979.5, title of invention: a simple and efficient autologous platelet-rich plasma extraction device and extraction method) used in the syringe, venous infusion set connecting tube, venous indwelling needle plastic card A device composed of an IV infusion set needle, the device described in this document can be used in the method of the present invention, and makes the method of the present invention beneficial in maintaining the cleanliness of the operation process.
出人意料地发现，本发明在研究梯度离心法进行血小板的浓缩和分离的过程中，获得了一种操作过程简单、收率高、富集的血浆中血小板浓度高的方法。Surprisingly, it is found that the present invention obtains a method with simple operation process, high yield and high concentration of platelets in the enriched plasma during the process of concentrating and separating platelets by gradient centrifugation.
具体实施方式Detailed ways
通过下面的实施例可以对本发明进行进一步的描述,然而，本发明的范围并不限于下述实施例。本领域的专业人员能够理解,在不背离本发明的精神和范围的前提下,可以对本发明进行各种变化和修饰。本发明对试验中所使用到的材料以及试验方法进行一般性和/或具体的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细的描述。The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention. The present invention provides general and/or specific descriptions of the materials and test methods used in the tests. While many of the materials and methods of manipulation which are employed for the purposes of the invention are well known in the art, the invention is described here in as much detail as possible.
以下提供了本发明操作方法的一般流程，在下文实施例中如未另外注明，使用下面一般流程的操作条件：The general flow of the operating method of the present invention is provided below, and if not otherwise specified in the following examples, the operating conditions of the following general flow are used:
(1)把患者的全血采集到含有抗凝剂的容器里，容器是采血袋。抗凝剂可以使用乙二胺四乙酸。抗凝剂和全血的比例为1：9。(1) Collect the patient's whole blood into a container containing anticoagulant, the container It's a blood collection bag. Anticoagulant ethylenediaminetetraacetic acid can be used. The ratio of anticoagulant to whole blood is 1:9.
(2)将血液和抗凝剂充分混匀，避免出现凝血。提取100微升以计算血小板数量。(2) Fully mix the blood and anticoagulant to avoid coagulation. Extract 100 microliters to count platelets.
(3)把血液分配到离心管(15毫升)。(3) Dispense the blood into centrifuge tubes (15 ml).
(4)把离心管放进离心机，以转速2400rpm离心4分钟。(4) Put the centrifuge tube into the centrifuge and centrifuge at 2400rpm for 4 minutes.
(5)离心后的血液应分为三层：最底层的是红细胞，最上层的是含血小板血浆，中间一层是Buffy Coat，含有血小板和白细胞。(5) The blood after centrifugation should be divided into three layers: the bottom layer is red blood cells, the top layer is platelet-containing plasma, and the middle layer is Buffy Coat, which contains platelets and white blood cells.
(6)利用移液器把最上层的血浆和大部分的buffy coat抽出并转移到新的离心管中，尽量避免抽取红细胞。提取100微升以计算血小板数量。(6) Use a pipette to extract the uppermost layer of plasma and most of the buffy coat and transfer them to a new centrifuge tube, avoiding extraction of red blood cells as much as possible. Extract 100 microliters to count platelets.
(7)把含有血浆和buffy coat混合物的离心管放进离心机，以转速1500rpm离心20分钟。(7) Put the centrifuge tube containing the mixture of plasma and buffy coat into the centrifuge, and centrifuge at 1500rpm for 20 minutes.
(8)把3/4的低浓度血小板血浆(Platelet Poor Plasma：PPP)去掉，利用剩下的1/4血浆重悬沉淀下来的血小板，获得富血小板血浆(PRP)。(8) Remove 3/4 of the low-concentration platelet plasma (Platelet Poor Plasma: PPP), and use the remaining 1/4 of the plasma to resuspend the precipitated platelets to obtain platelet-rich plasma (PRP).
(9)抽取100微升PRP以进行血小板计数。(9) Take 100 microliters of PRP for platelet count.
实施例1：提取富血小板血浆(PRP)Example 1: Extraction of platelet-rich plasma (PRP)
(1)分离前全血中血小板浓度：250×106/ml，全血体积为10ml。总血小板数量为2.5×109。(1) Platelet concentration in whole blood before separation: 250×106 /ml, the volume of whole blood is 10ml. The total platelet count was 2.5×109 .
(2)第一次离心后血浆中血小板浓度：570×106/ml(浓缩2.28倍)，血浆体积为4ml。总血小板数量为2.28×109。回收率为91.2%。(2) Platelet concentration in plasma after the first centrifugation: 570×106 /ml (concentrated 2.28 times), and the plasma volume is 4ml. The total platelet count was 2.28×109 . The recovery rate was 91.2%.
(3)第二次离心后血浆中(PRP)血小板浓度：1685×106/ml(浓缩6.74倍)，血浆体积为1.2ml，得到富血小板血浆。总血小板数量为2.02×109。最终回收率为80.8%。(3) Platelet concentration in plasma (PRP) after the second centrifugation: 1685×106 /ml (concentrated 6.74 times), plasma volume is 1.2ml, and platelet-rich plasma is obtained. The total platelet count was 2.02×109 . The final recovery rate was 80.8%.
实施例2：提取富血小板血浆(PRP)Example 2: Extraction of platelet-rich plasma (PRP)
使用乙二胺四乙酸二钠为抗凝剂，抗凝剂与全血的重量比为1：8。Disodium edetate was used as the anticoagulant, and the weight ratio of anticoagulant to whole blood was 1:8.
(1)分离前全血中血小板浓度：165×106/ml，全血体积为10ml。总血小板数量为1.65×109。(1) Platelet concentration in whole blood before separation: 165×106 /ml, whole blood volume is 10ml. The total platelet count was 1.65×109 .
(2)第一次离心后血浆中血小板浓度：444×106/ml(浓缩2.69倍)，血浆体积为3.5ml。总血小板数量为1.55×109。回收率为93.9%。(2) Platelet concentration in plasma after the first centrifugation: 444×106 /ml (concentrated 2.69 times), and the plasma volume is 3.5ml. The total platelet count was 1.55×109 . The recovery rate was 93.9%.
(3)第二次离心后血浆中(PRP)血小板浓度：1105×106/ml(浓缩6.70倍)，血浆体积为1.2ml，得到富血小板血浆。总血小板数量为1.33×109。最终回收率为80.6%。(3) Platelet concentration in plasma (PRP) after the second centrifugation: 1105×106 /ml (concentrated 6.70 times), plasma volume is 1.2ml, and platelet-rich plasma is obtained. The total platelet count was 1.33×109 . The final recovery rate was 80.6%.
实施例3：提取富血小板血浆(PRP)Example 3: Extraction of platelet-rich plasma (PRP)
使用枸橼酸葡萄糖为抗凝剂。Glucose citrate was used as an anticoagulant.
(1)分离前全血中血小板浓度：210×106/ml，全血体积为10ml。总血小板数量为2.1×109。(1) Platelet concentration in whole blood before separation: 210×106 /ml, whole blood volume is 10ml. The total platelet count was 2.1×109 .
(2)第一次离心后血浆中血小板浓度：542×106/ml(浓缩2.58倍)，血浆体积为3.5ml。总血小板数量为1.90×109。回收率为90.5%。(2) Platelet concentration in plasma after the first centrifugation: 542×106 /ml (concentrated 2.58 times), and the plasma volume is 3.5ml. The total platelet count was 1.90×109 . The recovery rate was 90.5%.
(3)第二次离心后血浆中(PRP)血小板浓度：1370×106/ml(浓缩6.52倍)，血浆体积为1.2ml，得到富血小板血浆。总血小板数量为1.64×109。最终回收率为78.1%。(3) Platelet concentration in plasma (PRP) after the second centrifugation: 1370×106 /ml (concentrated 6.52 times), plasma volume is 1.2ml, and platelet-rich plasma is obtained. The total platelet count was 1.64×109 . The final recovery rate was 78.1%.
实施例4：提取富血小板血浆(PRP)Embodiment 4: extract platelet-rich plasma (PRP)
抗凝剂与全血的重量比为1：10，第一次离心为2300rpm并5min，第二次离心为1600rpm并18min。The weight ratio of anticoagulant to whole blood is 1:10, the first centrifugation is 2300rpm and 5min, and the second centrifugation is 1600rpm and 18min.
(1)分离前全血中血小板浓度：195×106/ml，全血体积为10ml。总血小板数量为1.95×109。(1) Platelet concentration in whole blood before separation: 195×106 /ml, whole blood volume is 10ml. The total platelet count was 1.95×109 .
(2)第一次离心后血浆中血小板浓度：535×106/ml(浓缩2.74倍)，血浆体积为3.5ml。总血小板数量为1.87×109。回收率为95.9%。(2) Platelet concentration in plasma after the first centrifugation: 535×106 /ml (concentrated 2.74 times), and the plasma volume is 3.5ml. The total platelet count was 1.87×109 . The recovery rate was 95.9%.
(3)第二次离心后血浆中(PRP)血小板浓度：1235×106/ml(浓缩6.33倍)，血浆体积为1.2ml，得到富血小板血浆。总血小板数量为1.48×109。最终回收率为75.9%。(3) Platelet concentration in plasma (PRP) after the second centrifugation: 1235×106 /ml (concentrated 6.33 times), plasma volume is 1.2ml, and platelet-rich plasma is obtained. The total platelet count was 1.48×109 . The final recovery rate was 75.9%.
实施例5：提取富血小板血浆(PRP)Embodiment 5: extract platelet-rich plasma (PRP)
第一次离心为2500rpm并3min，第二次离心为1400rpm并20min。The first centrifugation is 2500rpm and 3min, and the second centrifugation is 1400rpm and 20min.
(1)分离前全血中血小板浓度：265×106/ml，全血体积为10ml。总血小板数量为2.65×109。(1) Platelet concentration in whole blood before separation: 265×106 /ml, whole blood volume is 10ml. The total platelet count was 2.65×109 .
(2)第一次离心后血浆中血小板浓度：612×106/ml(浓缩2.31倍)，血浆体积为4ml。总血小板数量为2.45×109。回收率为92.5%。(2) Platelet concentration in plasma after the first centrifugation: 612×106 /ml (concentrated 2.31 times), and the plasma volume is 4ml. The total platelet count was 2.45×109 . The recovery rate was 92.5%.
(3)第二次离心后血浆中(PRP)血小板浓度：1839×106/ml(浓缩6.94倍)，血浆体积为1.2ml，得到富血小板血浆。总血小板数量为2.21×109。最终回收率为83.4%。(3) Platelet concentration in plasma (PRP) after the second centrifugation: 1839×106 /ml (concentrated 6.94 times), the volume of plasma was 1.2ml, and platelet-rich plasma was obtained. The total platelet count was 2.21×109 . The final recovery rate was 83.4%.
比较例1：除了第一次离心转速改为2600rpm、3000rpm、3500rpm，并且这三种转速均安排3min、6min、10min时间外，其余操作条件均同实施例1。结果显示这样获得的9种富血小板血浆，它们的血小板最终回收率仅在47~63%之间，并且血小板的浓度是分离前全血中血小板浓度的1.8~3.7倍(在本发明中可称为最终富集倍数)。例如2600rpm并3min和2600rpm并6min两种情况下，血小板最终回收率分别为56%和61%，最终富集倍数分别为3.1和2.8。Comparative Example 1: Except that the first centrifugal speed was changed to 2600rpm, 3000rpm, and 3500rpm, and these three speeds were arranged for 3min, 6min, and 10min, the rest of the operating conditions were the same as in Example 1. The result shows that 9 kinds of platelet-rich plasmas obtained in this way, their platelet final recovery rate is only between 47～63%, and the concentration of platelet is 1.8～3.7 times of the platelet concentration in the whole blood before separation (in the present invention can be referred to as is the final enrichment factor). For example, in the two cases of 2600rpm and 3min and 2600rpm and 6min, the final recovery rates of platelets were 56% and 61% respectively, and the final enrichment factors were 3.1 and 2.8 respectively.
比较例2：除了第一次离心转速改为2100rpm、1700rpm、1400rpm，并且这三种转速均安排3min、6min、10min时间外，其余操作条件均同实施例1。结果显示这样获得的9种富血小板血浆，它们的血小板最终回收率仅在31~52%之间，并且血小板的浓度是分离前全血中血小板浓度的1.3~2.5倍。例如2100rpm并3min和2100rpm并6min两种情况下，血小板最终回收率分别为42%和47%，最终富集倍数分别为1.9和2.2。Comparative Example 2: Except that the first centrifuge speed was changed to 2100rpm, 1700rpm, and 1400rpm, and these three speeds were arranged for 3min, 6min, and 10min, the rest of the operating conditions were the same as in Example 1. The results showed that the final recovery rate of platelets in the 9 kinds of platelet-rich plasma obtained in this way was only between 31-52%, and the concentration of platelets was 1.3-2.5 times of that in whole blood before separation. For example, in the two cases of 2100rpm and 3min and 2100rpm and 6min, the final recovery rates of platelets were 42% and 47%, respectively, and the final enrichment factors were 1.9 and 2.2.
比较例3：除了第二次离心转速改为1800rpm、2200rpm、2600rpm，并且这三种转速均安排18min、20min、25min时间外，其余操作条件均同实施例1。结果显示这样获得的9种富血小板血浆，它们的血小板最终回收率仅在52~63%之间，并且血小板的浓度是分离前全血中血小板浓度的3.1~3.9倍。例如1800rpm并18min和1800rpm并20min两种情况下，血小板最终回收率分别为53%和57%，最终富集倍数分别为3.9和3.2。Comparative Example 3: Except that the rotational speed of the second centrifuge was changed to 1800rpm, 2200rpm, and 2600rpm, and these three rotational speeds were arranged for 18min, 20min, and 25min, the rest of the operating conditions were the same as in Example 1. The results showed that the final recovery rate of platelets in the 9 kinds of platelet-rich plasma obtained in this way was only between 52-63%, and the concentration of platelets was 3.1-3.9 times that of whole blood before separation. For example, in the two cases of 1800rpm and 18min and 1800rpm and 20min, the final recovery rates of platelets were 53% and 57% respectively, and the final enrichment factors were 3.9 and 3.2 respectively.
比较例4：除了第二次离心转速改为1300rpm、1000rpm、700rpm，并且这三种转速均安排18min、20min、25min时间外，其余操作条件均同实施例1。结果显示这样获得的9种富血小板血浆，它们的血小板最终回收率仅在33~46%之间，并且血小板的浓度是分离前全血中血小板浓度的1.9~3.1倍。例如1300rpm并18min和1300rpm并20min两种情况下，血小板最终回收率分别为43%和35%，最终富集倍数分别为2.9和2.4。Comparative Example 4: Except that the rotational speed of the second centrifuge was changed to 1300rpm, 1000rpm, and 700rpm, and these three rotational speeds were arranged for 18min, 20min, and 25min, the rest of the operating conditions were the same as in Example 1. The results showed that the final recovery rate of platelets in the 9 kinds of platelet-rich plasma obtained in this way was only between 33-46%, and the concentration of platelets was 1.9-3.1 times of that in whole blood before separation. For example, in the two cases of 1300rpm and 18min and 1300rpm and 20min, the final recovery rates of platelets were 43% and 35% respectively, and the final enrichment factors were 2.9 and 2.4 respectively.
比较例5：第一次离心改为2300rpm并4min、或者2500rpm并4min，并且第二次离心改为1400rpm并20min、或者1600rpm并20min，其余操作条件均同实施例1。结果显示这样获得的4种富血小板血浆，它们的血小板最终回收率仅在48~61%之间，并且血小板的浓度是分离前全血中血小板浓度的3.2~4.2倍。例如第一次离心为2300rpm并4min、第二次离心为1600rpm并20min，血小板最终回收率为51%，最终富集倍数分别为3.7。Comparative Example 5: The first centrifugation was changed to 2300rpm and 4min, or 2500rpm and 4min, and the second centrifugation was changed to 1400rpm and 20min, or 1600rpm and 20min, and the rest of the operating conditions were the same as in Example 1. The results showed that the final platelet recovery rate of the four kinds of platelet-rich plasma obtained in this way was only between 48-61%, and the platelet concentration was 3.2-4.2 times that of the whole blood before separation. For example, the first centrifugation is 2300rpm and 4min, and the second centrifugation is 1600rpm and 20min, the final recovery rate of platelets is 51%, and the final enrichment factor is 3.7 respectively.
此外，参考CN 102078644A实施例1的操作，血小板最终回收率为47%，最终富集倍数分别为3.45。In addition, referring to the operation in Example 1 of CN 102078644A, the final recovery rate of platelets was 47%, and the final enrichment factor was 3.45.
然而，本发明以上结果显示，本发明方法在第一次和第二次离心后的平均血小板回收率分别为：92.8%和79.8%，分离后的PRP中血小板的浓度可以达到全血的6-7倍。表明本发明所用的离心的时间和转速的设定能有效分离和回收大部分全血中的血小板。However, the above results of the present invention show that the average platelet recovery rate of the inventive method after the first and second centrifugation is respectively: 92.8% and 79.8%, and the concentration of platelets in the separated PRP can reach 6-6% of whole blood. 7 times. It shows that the setting of centrifugation time and rotational speed used in the present invention can effectively separate and recover most of the platelets in the whole blood.
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| CN201410570704.2ACN104383726B (en) | 2012-07-30 | 2012-07-30 | The extracting method of platelet rich plasma and the platelet rich plasma of extraction |
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