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首页> 外文会议>Conference on biochemical and molecular engineering >THE SEPARATION OF RED BLOOD CELLS BASED SOLELY ON INTRINSIC MAGNETIZATION: CLINICAL AND COMMERCIAL IMPLICATIONS
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THE SEPARATION OF RED BLOOD CELLS BASED SOLELY ON INTRINSIC MAGNETIZATION: CLINICAL AND COMMERCIAL IMPLICATIONS

机译：仅基于内在磁化作用的红色血细胞分离：临床和商业意义

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摘要

A rough estimate puts the cell isolation market at approximately $ 6 billion a year worldwide. One of the key commercial technologies uses antibodies conjugated to magnetic micro and nanoparticles (i.e Dynal beads or Miltenyi MACS systems). While clearly effective, whenever antibodies are used, whether conjugated to magnetic particles, or fluorescent molecules (such as used in FACS systems), there is always the issue of the sensitivity and specificity of the antibody for the targeted cell(s). This "issue", amongst others, is the motivator for "label free" identification and separation technology. Removal of human red blood cells, hRBCs, from a blood or bone marrow sample for diagnostic, or therapeutic applications is a fundamental laboratory practice/procedure. While difficult to obtain precise numbers, it has been suggested that greater than a billion blood draws are conducted in the US each year. While for a majority of these blood draws an evaluation of the RBCs is an important part, there is still a very large number of tests that focus on the remaining blood components after the RBCs have been removed. While not nearly as common as a blood draw, more than 18,000 bone marrow or umbilical cord blood transplants were performed in the US in 2013. In the case of bone marrow transplants, the RBCs need to be removed prior to transfusion or cryopreservation, regardless of whether the donor and patient's tissues match. Viewed from a mechanistic perspective, there are three primary methodologies to remove human RBCs, hRBCs, from a blood draw: 1) RBC lysis, 2) immunological based separation in which a RBC is bound with an affinity ligand which facilitates RBC removal, or 3) separation of the RBC from the nucleated cells based on density differences. The two most commonly used methods are the density difference methods with or without hydrophilic polysaccharide addition (e.g Ficoll density gradient based centrifugation, DGC,). When blood samples are only used for further analysis, the condition and the content of the sample after the RBC removal is only important with respect to how it affects the subsequent analysis; however, when the RBC depleted sample is destined for transfusion into a patient, significantly higher standards are required. We previously compared RBC removal using the Ficoll-based DGC to lysis protocols. Using either method would remove more than 99% of RBCs; however the average recovery of the spiked cancer cells was 73 and 89% for the Ficoll and RBC lysis, respectively. Poor recovery of targeted cells, such as hematopoietic stem cells, in the initial RBC depletion step is a problem in the bone marrow transplant/regenerative medicine community. In fact, several reports indicate that the recovery of nucleated cells from bone marrow, BMNCs, using Ficoll-based DGC, can be as low as 15-30%. Complementary to these reports, two recent papers suggest that cells with high regenerative potential, such as very small embryonic-like stem cells, VSELs and mesenchymal stromal cells are depleted with DGC. Finally, there are suggestions that Ficoll DGC can impair receptor function of the recovered cells. It is well established that deoxygenated RBCs are weakly paramagnetic; initially reported by Linus Pauling and coworkers in 1936. Melville and co-workers demonstrated in the mid 1970's that RBCs can be captured using a ferromagnetic wire mesh when the cells are reduced (chemical turned into a state equivalent to the deoxy-state). More recently, we have demonstrated that RBCs can be captured in HGMS systems (i.e. Miltenyi Biotec MACS columns), magnetically deposited on slides, deposited on the wall of a channel, and continuous removed using a flow through separation system. While these studies demonstrate theoretically, and experimentally, that it is possible to separate RBCs based on intrinsic magnetization, the throughputs in these studies are orders of magnitude lower than needed to practically remove RBCs from a typical blood draw. In this presentation we will present our latest systems which we suggest can increase the throughputs by orders of magnitude which presents the potential for magnetic separation of RBCs to become a practical alternative to the currently used approaches.
机译：粗略估计，全球细胞隔离市场每年约为60亿美元。关键的商业技术之一是使用偶联到磁性微粒和纳米颗粒上的抗体（即Dynal磁珠或Miltenyi MACS系统）。尽管明显有效，但是无论何时使用抗体，无论是缀合至磁性颗粒还是荧光分子（例如用于FACS系统中的抗体），始终存在抗体对靶细胞的敏感性和特异性的问题。除其他外，该“问题”是“无标签”识别和分离技术的推动力。从血液或骨髓样本中去除人红细胞，hRBC以用于诊断或治疗应用是一项基本的实验室操作/程序。尽管很难获得准确的数字，但有人建议每年在美国进行的抽血超过十亿次。尽管对这些血液中的大多数来说，对RBC的评估是很重要的一部分，但仍然有大量测试着眼于去除RBC后剩余的血液成分。尽管不像抽血那样普遍，但2013年在美国进行了超过18,000例骨髓或脐带血移植。就骨髓移植而言，无论输血或冷冻保存，都应先去除红细胞，无论供体和患者的组织是否匹配。从机理的角度来看，有三种主要的方法可从抽血中去除人的RBC，hRBC：1）RBC裂解; 2）基于免疫学的分离，其中RBC与促进RBC去除的亲和配体结合，或3 ）根据密度差异从有核细胞中分离出RBC。两种最常用的方法是添加或不添加亲水性多糖的密度差法（例如基于Ficoll密度梯度的离心DGC）。如果仅将血液样本用于进一步分析，则去除红细胞后的状况和样本含量仅对如何影响后续分析很重要;但是，当将RBC耗尽的样品用于输血时，则需要更高的标准。我们以前将使用基于Ficoll的DGC的RBC去除与裂解方案进行了比较。无论使用哪种方法，都将去除超过99％的RBC。然而，Ficoll和RBC裂解的加标癌细胞的平均回收率分别为73％和89％。在最初的RBC消耗步骤中，诸如造血干细胞之类的靶细胞恢复不良，是骨髓移植/再生医学界的一个问题。实际上，一些报告表明，使用基于Ficoll的DGC从骨髓BMNC中回收有核细胞的比例可能低至15％至30％。作为这些报告的补充，最近的两篇论文表明，具有高再生潜力的细胞，例如非常小的胚胎样干细胞，VSEL和间充质基质细胞，都被DGC消耗掉了。最后，有人提出Ficoll DGC可能损害回收细胞的受体功能。众所周知，脱氧的RBC具有弱顺磁性。最初是由Linus Pauling及其同事在1936年报道的。Melville及其同事在1970年代中期证明，当细胞被还原时（化学物质变成与脱氧状态相当的状态），可以使用铁磁丝网捕获RBC。最近，我们证明了RBC可以在HGMS系统（即Miltenyi Biotec MACS色谱柱）中捕获，磁性沉积在载玻片上，沉积在通道壁上，并使用流通分离系统连续去除。尽管这些研究从理论上和实验上证明了可以基于固有磁化强度分离RBC的可能性，但这些研究中的通量比从典型的抽血中实际去除RBC所需的通量要低几个数量级。在本演讲中，我们将介绍我们的最新系统，我们建议将这些系统的吞吐率提高几个数量级，这表明RBC的磁选潜力有可能成为当前使用方法的一种实用替代方案。

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