CN1238083C - 制造富含血小板的血浆和/或血小板浓缩液的方法和设备 - Google Patents

制造富含血小板的血浆和/或血小板浓缩液的方法和设备 Download PDF

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CN1238083C
CN1238083C CNB008005648A CN00800564A CN1238083C CN 1238083 C CN1238083 C CN 1238083C CN B008005648 A CNB008005648 A CN B008005648A CN 00800564 A CN00800564 A CN 00800564A CN 1238083 C CN1238083 C CN 1238083C
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CN1300233A (zh
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卢·布拉塞蒂
舍温·V·凯维
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Abstract

通过将全血放置到具有两个室(6、8)的一次性无菌容器的第一室(6)内制得富含血小板的血浆和/或血小板浓缩液的方法。对一次性无菌容器进行第一次离心处理从而分离红细胞(36)，把形成的富含血小板的血浆的上清液(38)倾倒到第二室(8)内。对该一次性无菌容器进行第二次离心处理以形成血小板浓缩液。去除第二室(8)内一定量的血小板贪乏的血浆上清液，并且将血小板重新悬浮在剩余的血浆内。第二室(8)可以装有抗凝血剂(34)，以防止血小板凝集。

Description

制造富含血小板的血浆和/或血小板浓缩液的方法和设备
技术领域
本发明涉及制造富含血小板的血浆或者血小板浓缩液的方法和设备，特别是用于分离血小板和血浆以及用于按照选定的比例混合血小板和血浆从而提供富含血小板的血浆或具有选定浓度的血小板浓缩液的自动化高效方法。
背景技术
一般的用于制造富含血小板的血浆(PRP)的方法涉及对全血进行“轻度”离心处理。血小板浓缩液(PC)则通过对PRP进行第二次离心处理得到。
富含血小板的血浆PRP或血小板浓缩液PC中的血小板含有颗粒，这些颗粒含有生长因子(例如PDGF，TGF-β和其他)，这些生长因子有助于加速血管生成(伤口愈合)和骨头生成(骨头生长)。当与凝血酶一起使用时，PRP/PC还可以作为辅助手段来控制出血(止血)、愈合伤口以及作为输送药物和/或生物制剂的载体。另外，通过与PRP/PC结合，添加或者不添加凝血酶，某些有机材料，例如骨粉的处理性能可以得到极大改善。这种组合也使得有机材料更加紧固地固定到例如整形缺陷部位内。PRP/PC和凝血酶(例如用于止血和伤口愈合)的某些性质类似于纤维蛋白胶的性质，其区别在于：纤维蛋白胶具有更大的粘性，因为它的纤维蛋白原的浓度在基准水平以上。
制造PC的典型方法涉及对血袋中所收集的全血进行离心处理，从而将PRP与红细胞分开。然后，PRP从第一血袋快速转移到第二血袋内，再进行一次离心处理，从而形成血小板浓缩液(“小球”)PC和血小板贪乏的血浆上清液(PPP)。PPP的大部分被快速转移到第三血袋中，而把浓缩的血小板和少量的PPP留在第二血袋中，用于重新悬浮浓缩的血小板。这种方法的典型的血小板回收率只有45％，因此该方法用于针对治疗点使用时过于复杂，因此不能进行自体同源血液产品的针对治疗点使用的生产。
美国专利5707331(wells)中公开了一种用于从血浆生产自体同源的纤维蛋白原的自动化系统。该专利所公开的系统通过对全血进行自动化处理，将全血离心处理得到血浆成分，对离心处理后的血浆成分进一步进行物理化学沉淀处理，并且进一步进行离心处理而形成纤维蛋白原成分。回收得到纤维蛋白原，当其与凝血酶一起使用时得到一种纤维密封剂。
从少量的全血中根据需要生产PRP/PC的能力将会大大提高PRP/PC的临床实用性，而且自体同源的PRP/PC的可利用性会消除对同源的PRP/PC的需要，因为同源的PRP/PC可能存在传播某种人类疾病的危险。而且，通常需要提供给定浓度的PRP/PC来实现某种特定的治疗效果。但是，公知的用于生产PRP/PC的方法费时、效率低，从而不适合于用少量的全血进行生产。
因此，本发明的目的就是提供一种方法和设备，用于有效地把少量的全血在治疗点处理成具有所需浓度和临床所需的PRP或者PC。
技术方案
根据本发明，通过一个自动化的方法可以容易地制造少量的PRP或者PC。该方法最好通过例如美国专利US5707331(Wells)中的离心机来实现。该美国专利US5707331中的离心机装有一个一次性容器或者一次性处理管(PD)，具有两个室。在本发明的方法中，全血首先放置到PD的一个室中。然后对该离心机进行操作从而导致红细胞沉淀到一个室的底部而形成PRP上清液。停止/减弱离心处理，或者通过重力或者离心输送方法使PRP倾倒到第二室内。
之后，通过重新启动/加速离心机，对第二室内的PRP进行第二次离心处理。然后停止离心处理，从而(1)在第一室内得到红细胞；(2)在第二室的底部得到血小板(PC)；(3)在第二室内以上清液的形式得到血小板贪乏的血浆(PPP)。上述的离心机操作过程最好是自动执行的。
通过得到规定量的血浆上清液和重新悬浮血小板，操作者可以制造所需浓度的PRP/PC。
在一个最佳实施例中，操作者将一个固定到注射器上的钝头套管插入到第二室内，取出一定量的血浆，而留下已知一定量的血浆。然后把固定到注射器上的第二个钝头套管插入到第二室，其中的剩余已知量的血浆用于重新悬浮，得到增加了血小板浓度的PRP/PC。
也可以用其它方法复原血小板和血浆。例如，在自动化步骤完成后，使用者可以通过倾斜一次性容器而从第二室倾倒出血浆，从而导致一定量的血浆流回到第一室内，而留下一定量的血浆在第二室内。混合剩余的血浆和血小板后复原血小板和血浆。
在一个例子中，获取病人全血血样，一般该血样的血小板浓度为220×103/ul。根据一个典型的血小板回收率60％和典型的血液处理量50ml，重新将PC悬浮在5ml的PPP中，所提供的PRP的血小板浓度为1320×103/ul，血小板浓缩液的浓度增加了6倍。
附图简述
图1表示根据本发明的一次性处理管和离心机；
图2是图1中的处理管的侧视图，局部剖视；
图3a到3f是图2中的处理管的示意剖面图，表示根据本发明进行离心处理过程中处理管的不同方向。
优选实施例详述
图1示意表示了根据本发明的一个离心机2和一个一次性处理管(PD)4。优选的离心机为美国专利US5707331(Wells)中所述的系统，该系统可以下面结合图3所述的方式而进行编程操作。可以理解，离心机2的转子可以设计为可以同时容纳一个或者多个处理管PD4。在优选实施例中，该离心机容纳一个或者两个处理管PD。当只使用一个处理管时，在该装填后的处理管对面放置一个平衡重。
根据本发明的处理管如图2所示，并参见美国专利US5707331。该处理管PD最好由模制的塑料制成，并且包括至少两个室6、8。这两个室由一连接它们的桥接件10在顶部最好相联接。这两个室由一个盖12封闭，该盖12用于保持流体通道的无菌状态。
盖12包括凸起18和20，这两个凸起分别具有开口22和24，用于允许进入到室的内部。室6包括一个搁板26，用于协助从细胞成分中分离PRP，下面进行详细描述。室6还包括一个空心管28，该空心管28从开口22穿过搁板26延伸，从而使得液体可以进入到室6内。搁板的周边允许搁板26下方的血浆向上流动。
下面参照图3a到3f说明按照本发明工艺方法离心机2的操作。在本工艺方法的第一步，将量好量的生理液体32，比如全血，装到PD4的室6中，将一些(如1～5ml，优选2ml)抗凝血剂34，优选ACD-A，加到室8中。之后，如图3b所示，对PD进行离心作用。这就将生理液体中的红细胞36等重组分与PRP38类的上清液分离。ACD-A34仍留在室8中。
图3b所示的第一次离心处理使得红细胞与PRP分离，但并没有将血小板与血浆的剩余物有效分离。在优选实施例中，该第一次离心处理在约1200G(约3600RPM)下进行了约2分钟。
为清楚起见，图3a到3f没有表示搁板26，但是需要指出的是，在优选实施例中，搁板尽可能靠近两个分离成分，亦即红细胞36和血浆38之间的边界。用于完成这种功能的优选的方法是确定病人血液中红细胞的浓度(例如用血球容积计)从而使得所提供的血液中的红细胞能够充满搁板下方。作为优选的方式，室6的额定容积设计为容纳50ml的病人血液。这个量可以在设备的操作过程中根据血球容积计进行调节，本申请人已经发现，所需全血的容积为40～60ml。
在红细胞被离心分离后，处理管PD锁定在重力排出位置，如图3c所示。这一点在美国专利US5707331中也进行了描述，并且这种分离最好由一个电磁铁的电激活作用来完成，该电磁铁移动一个锁定盘从而使得该锁定盘与一个盛装处理管PD的保持器啮合。当处理管处于该位置上时，室6内的PRP38在重力作用下流到室8内。例如，25ml的PRP转移到室8内。PRP38在通过流体通道16流到室6内时，还与事先放置到室8内的ACD-A 34混合。
在图3c的流出步骤中，通常需要继续以较低的速度例如60RPM旋转转子，从而提供较小的离心力而将红细胞36保持在室6内。
如图3d所示，对离心机进行加速从而对PRP38进行第二次离心处理。该第二次离心处理将血小板40与PPP上清液42分离。在优选实施例中，第二次离心处理进行约8分钟，转速大约为1000G(约3000RPM)。
可以理解，第一次和第二次离心处理的具体的转速可以改变。例如，第二次离心处理可以是强力旋转(hard spin)。并且，所公开的优选转速是针对转子最大半径为4英寸(从轴线到室的底部所测量的旋转半径)的离心机。其它尺寸的离心机需要不同的转速。
ACD-A放置到室8内，用于减少血小板的凝集。已经发现，在第二室内的抗凝血剂可以减少血小板的聚集，从而减少进行离心处理的整个时间。
本发明工艺方法的下一步如图3e所示。在该步骤中，离心机停止，而允许处理管PD处于直立方向，其中，红细胞36保持在室6内，血小板40保持在室8的底部，而PPP 42则作为上清液保持在室8中。一个具有钝头套管46的皮下注射器44用于抽出预定量的PPP。这可以通过将钝头套管穿过开口24而插入到预定深度来完成。操作者可以手动调节其深度，或者如图3e所示，可以在钝头套管上设置一个高度调节尺48，在所需深度时防止钝头套管继续插入。高度调节尺可以具有任何形式，优选为一个空心管，该空心管安装到钝头套管外并与注射器的底部啮合。另外，一个具有多个不同长度的调节尺的工具包也可以用于帮助操作者选择抽出不同预定量的PPP。
另外，抽出给定量的PPP也可以通过将一部分血浆倒回到室6中来实现，这可以通过手动方式，或者通过利用美国专利5707331中所述的多倒回特性的离心机进行离心式转移。
从如图3e所示的方法继续进行，在钝头套管46插入到所需深度后操作注射器从而抽出给定量的PPP，该PPP然后用于其它目的例如止血。
如图3f所示，血小板40然后重新悬浮在剩余的PPP中从而形成具有所需血小板浓度的PRP/PC50，其浓度比原始的上清液38的高几倍。这种增加了浓度的PRP/PC然后用于现有技术中已知的各种用途。
显然，本领域的普通技术人员可以在权利要求所要求的保护范围内修改本发明。

Claims (14)

1.一种用于制造具有选定组分的生理产品的方法，包括下列步骤：
将具有多种组分的生理液体放置到具有水平隔开的第一室和第二室的刚性无菌容器的第一室内；
对所述的生理液体进行离心处理，以从第一上清液分离至少一种所述的组分；
将所述的第一上清液倾倒到所述的第二室；
对所述的第一上清液进行离心处理，以从第二上清液分离第二种所述的组分；
从所述的第二室内取出预定量的所述的第二上清液，使得第二上清液的剩余物保留在第二室内；以及
在第二室内将所述的第二种组分重新悬浮在所述的第二上清液的剩余物内。
2.根据权利要求1所述的方法，还包括将抗凝血剂放到所述的第二室内的步骤。
3.根据权利要求1所述的方法，其中，所述的生理液体是血液。
4.根据权利要求3所述的方法，其中，所述的生理产品是富含血小板的血浆，所述的对生理液体进行离心处理的步骤包括对所述的血液进行大约2分钟的第一次离心处理的步骤。
5.根据权利要求4所述的方法，其中，所述的对第一上清液进行离心处理的步骤包括对所述的富含血小板的血浆进行大约8分钟的第二次离心处理的步骤。
6.根据权利要求1所述的方法，其中，所述取出预定量的第二上清液的步骤包括：将所述套管插入所述第二室内，然后通过所述套管取出所述预定量的第二上清液。
7.根据权利要求6所述的方法，还包括：将高度调节尺连接到所述套管以确定所述套管插入所述第二室深度的步骤。
8.根据权利要求7所述的方法，其中，所述的预定量是所述第二室内初始的流体量与由所述高度调节尺高度限定的所述第二室内所需流体量之间的差值。
9.根据权利要求1所述的方法，其中，所述的预定量是在所述第二室内超过所述第二室所需流体量的所述第二上清液量。
10.一种在治疗中制备富含血小板的血浆的方法，该方法包括：将血液放置到具有第一室和第二室的无菌容器的第一室内，其中，至少所述第二室是刚性的并具有确定的容量；对所述的容器和血液进行离心处理以获取包含有富含血小板的血浆的上清液；将所述富含血小板的血浆从所述第一室输送到第二室；对所述富含血小板的血浆进行离心处理以获取分离的血小板和血小板含量低的血浆；从所述第二室中取出部分所述血小板含量低的血浆并在所述第二室内保留剩余部分的所述血小板含量低的血浆和所述分离的血小板；在所述剩余部分血小板含量低的血浆中悬浮所述分离的血小板以获得所述富含血小板血浆。
11.根据权利要求10所述的方法，还包括将抗凝血剂放置在所述第二室内的步骤。
12.根据权利要求10所述的方法，其中所述取出的步骤包括把套管插入所述第二室内然后抽出所述血小板含量低的血浆。
13.一种生产生理产品的药箱，包括：
一个无菌容器，具有一个第一室和一个第二室，其中所述两个室彼此相连以使流体能够在两个室之间流动并且所述容器通过套管设置有通向所述第二室的无菌通道；
所述的套管；和
一个或多个限制所述套管插入所述第二室内深度的高度调节尺，
其中，所述高度调节尺包括一个装配在所述套管上方的中空管。
14.根据权利要求13所述的药箱，它包括至少两个长度不同的中空管。
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