CN115633679A - 富血小板血浆的分离保存液及分离方法 - Google Patents
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Abstract
本发明提供了一种富血小板血浆的分离保存液及分离方法,该分离保存液包括:2.4‑2.7g枸橼酸三钠、0.30‑0.33g枸橼酸、2.2‑2.7g葡萄糖、0.45‑0.52g重组人白蛋白、0.19‑0.22g磷酸二氢钠和100mL蒸馏水;分离方法包括:抽取血液后测血常规,记录血小板数量后,接着加入PRP的分离保存液;室温下离心;吸取上清及部分中间层白膜,室温下再次离心;弃去部分上清,保留沉淀物及血清,重悬,并复测血小板数量;本发明的分离保存液中不仅加入了枸橼酸和枸橼酸三钠,而且还采用了低速离心法,从而获得血小板浓度较高的富血小板血浆,经过临床反复验证,获得血小板的浓度比较稳定。
Description
技术领域
本发明属于血小板分离技术领域,具体涉及一种富血小板血浆的分离保存液及分离方法。
背景技术
富血小板血浆(PRP)治疗在临床的运用范围很广,主要是针对难以愈合的创面,感染严重的创面,骨折断端不愈合,慢性膝关节磨损,薄型子宫内膜修复等情况。现在医美方面的应用也非常广泛,可以用于脱发的治疗,有研究表明经过周期性的头皮注射,以改善局部的血液循环,促使休止期毛囊再次生长出毛发。富血小板血浆也可以用于面部水光针的注射,以改善暗沉皮肤的状态,修复痘坑痘痕,同时将它和自己颗粒脂肪混合之后可以提升颗粒脂肪的成活率。所以目前血小板血浆的运用范围广,那么安全高效的血小板血浆的分离在临床中被迫切需求。
目前血小板的获得方法分为两种,一种是分离液体外离心获得(募集血小板纯度低),另外一种通过单采血小板,体外循环获得(有创性,但获得的血小板浓度高)。
前期采用市场常见的血小板分离液(调制后的Tyrode’s缓冲液(136.9mM NaCl,12.1mM NaHCO3,2.6mM KCl,5.5mM glucose,and 10mM HEPES,pH分别调整为6.5和7.4)。此配方仅能满足粗略分离血小板,并不能在分离后继续保存血小板,并且易间接导致血小板激活。且高浓度的K+回输存在一定的临床风险,且患者局部疼痛感明显。
K+通路会影响钙流,可能导致血小板激活。实验研究发现,抑制K+通道后,血小板激活,成栓都减少[1]。
参考文献:
[1]Fan,C.,Yang,X.,Wang,W.W.,Wang,J.,Li,W.,Guo,M.,Huang,S.,Wang,Z.,&Liu,K.(2020).Role of Kv1.3 Channels in Platelet Functions and ThrombusFormation.Arteriosclerosis,thrombosis,and vascular biology,40(10),2360–2375.https://doi.org/10.1161/ATVBAHA.120.314278.
在采用市场现有的分离液和分离方法时,通常获得的血小板浓度较低,为了获得较高浓度的富血小板血浆,只能抽取患者更多的血液。
发明内容
针对现有技术中的不足,本发明的目的是提供一种富血小板血浆的分离保存液及分离方法。
为达到上述目的,本发明的解决方案是:
目的之一,本发明提供了一种PRP的分离保存液,其包括如下组分:
其中,枸橼酸和枸橼酸三钠起到抗凝的作用,并且通过螯合血液中的钙离子,防止血小板的提前激活,则可以保证血小板的活性以及不被激活,分离的PRP在放置24h后检测,仍然能保持较高的浓度。
葡萄糖和重组人白蛋白起到营养血小板的作用。
磷酸二氢钠主要作用是防止血液凝固。
目的之二,本发明提供了一种上述的PRP的分离保存液的分离方法,其包括如下步骤:
(1)、抽取血液后测血常规,记录血小板数量后,接着加入PRP的分离保存液;
(2)、室温下离心;
(3)、吸取上清及部分中间层白膜,室温下再次离心;
(4)、弃去部分上清,保留沉淀物及血清,重悬,并复测血小板数量。
优选地,步骤(1)中,PRP的分离保存液包括如下组分:
优选地,步骤(2)中,离心的转速可以为70-100rpm,优选为70rpm,低速分离可以匀速地分离红细胞和血小板,最大程度地减少了血小板的耗损;离心的时间可以为35-40min,优选为40min,不会出现分离不完全的现象,高于40min增加了临床工作的时间。
优选地,步骤(3)中,再次离心的转速可以为1000-1500rpm,优选为1500rpm,根据血小板的体积,1500rpm为最优;离心的时间可以为10-15min,优选为15min。
由于采用上述方案,本发明的有益效果是:
第一、本发明的分离保存液中不仅加入了枸橼酸和枸橼酸三钠,而且还采用了低速离心法,从而获得血小板浓度较高的PRP,经过临床反复验证,获得血小板的浓度比较稳定。
第二、本发明的分离液成分简单,无有毒有害物质,分离液需求量少,分离的PRP中残留分离液量少,且血小板浓度高;另外,分离液中不含K+,降低了临床回输的风险,降低回输的局部疼痛感提升了患者的舒适度。
附图说明
图1为现有技术的传统PRP分离结果示意图。
图2为本发明实施例的PRP分离结果示意图。
具体实施方式
本发明提供了一种PRP的分离保存液及分离方法。
下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例:
本实施例的PRP的分离保存液的分离方法包括如下步骤:
(1)、采用肝素抗凝管抽取20mL血液后测血常规,记录血小板数量后,接着加入4mL的PRP的分离保存液;
(2)、室温下离心,离心的转速为70rpm,采用升速0,降速0的最低加速离心方式,离心的时间为40min;
(3)、吸取上清及部分中间层白膜,室温下再次离心,再次离心的转速为1500rpm,离心的时间为15min;
(4)、弃去部分上清,保留沉淀物及1mL血清,重悬,即获得PRP,并复测血小板数量。
其中,步骤(1)中,PRP的分离保存液包括如下组分:
采用现有技术中的PRP分离方法,分离前后血小板浓度对比如图1和表1:
表1
由表1可知,传统法的分离倍数为3-6.5倍。
采用本发明实施例的PRP分离方法,分离前后血小板浓度对比如图2和表2:
表2
由表2可知,本实施例的的分离倍数平均达到10倍以上。因此,本实施例的4mL分离保存液,加入20mL患者血液,通过低速离心法,即可以获得血小板浓度较高的PRP,经过临床反复验证,获得血小板的浓度比较稳定,浓缩倍数为8-15倍,甚至优于单采血小板。
上述对实施例的描述是为了便于该技术领域的普通技术人员能理解和使用本发明。熟悉本领域技术人员显然可以容易的对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中,而不必经过创造性的劳动。因此,本发明不限于上述实施例。本领域技术人员根据本发明的原理,不脱离本发明的范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (5)
2.一种如权利要求1所述的富血小板血浆的分离保存液的分离方法,其特征在于:其包括如下步骤:
(1)、抽取血液后测血常规,记录血小板数量后,接着加入富血小板血浆的分离保存液;
(2)、室温下离心;
(3)、吸取上清及部分中间层白膜,室温下再次离心;
(4)、弃去部分上清,保留沉淀物及血清,重悬,并复测血小板数量。
4.根据权利要求2所述的分离方法,其特征在于:步骤(2)中,所述离心的转速为70-100rpm,离心的时间为35-40min。
5.根据权利要求2所述的分离方法,其特征在于:步骤(3)中,所述再次离心的转速为1000-1500rpm,离心的时间为10-15min。
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CN102755770A (zh) * | 2012-07-30 | 2012-10-31 | 博雅干细胞科技有限公司 | 富血小板血浆的提取方法和提取的富血小板血浆 |
CN104307208A (zh) * | 2014-09-28 | 2015-01-28 | 浙江中医药大学 | 一种富集纯化血小板的方法 |
CN107617236A (zh) * | 2017-09-28 | 2018-01-23 | 安徽信灵检验医学科技有限公司 | 一种血小板富集液 |
CN111407778A (zh) * | 2019-11-20 | 2020-07-14 | 广东先康达生物科技有限公司 | 一种富含血小板血浆的制备方法 |
CN113322232A (zh) * | 2021-04-29 | 2021-08-31 | 广东先康达生物科技有限公司 | 富含血小板血浆的制备方法 |
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CN102755770A (zh) * | 2012-07-30 | 2012-10-31 | 博雅干细胞科技有限公司 | 富血小板血浆的提取方法和提取的富血小板血浆 |
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CN113322232A (zh) * | 2021-04-29 | 2021-08-31 | 广东先康达生物科技有限公司 | 富含血小板血浆的制备方法 |
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